Modified subtilisins and detergent compositions containing same

ABSTRACT

Enzymes produced by mutating the genes for a number of subtilisin proteases and expressing the mutated genes in suitable hosts are presented. 
     The enzymes exhibit improved wash performance in comparison to their wild type parent enzymes. 
     The enzymes are well-suited for use in detergent compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.08/815,775 filed on Mar. 12, 1997 now U.S. Pat. No. 6,197,567, which isa continuation of U.S. application Ser. No. 08/484,190 filed on Jun. 7,1995 now U.S. Pat. No. 5,665,587, which is a continuation of U.S.application Ser. No. 07/776,115 filed on Oct. 15, 1991 now abandoned,which is a continuation-in-part of U.S. application Ser. No. 07/544,003filed on Jun. 26, 1990 now abandoned, which is a continuation-in-part ofU.S. application Ser. No. 07/516,026 filed on Apr. 26, 1990 nowabandoned, and claims priority under 35 U.S.C. 119 of Danish applicationSer. No. 3169/89 filed on Jun. 26, 1989, and Great Britain applicationNos. 8914604.7 filed on Jun. 26, 1989, and 8915660.8 filed on Jul. 7,1989, the contents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to novel mutant enzymes or enzyme variants usefulin formulating detergent compositions in exhibiting improved washperformance, cleaning and detergent compositions containing saidenzymes, mutated genes coding for the expression of said enzymes wheninserted in a suitable host cell or organism and methods of selectingthe amino acid residues to be changed in a parent enzyme in order toperform better in a given wash liquor under specified conditions.

BACKGROUND OF THE INVENTION

In the detergent industry, enzymes have been implemented in washingformulations for more than 20 years. Enzymes used in such formulationscomprise proteases, lipases, amylases, cellulases, as well as otherenzymes, or mixtures thereof. Commercially, proteases are mostimportant.

Although proteases have been used in the detergent industry for morethan 20 years, it is still not exactly known which physical or chemicalcharacteristics are responsible for a good washing performance orability of a protease.

The currently used proteases have been found by isolating proteases fromnature and testing them in detergent formulations.

Bacillus Proteases

Enzymes cleaving the amide linkages in protein substrates are classifiedas proteases, or (interchangeably) peptidases (see Walsh, 1979,Enzymatic Reaction Mechanisms. W.H. Freeman and Company, San Francisco,Chapter 3). Bacteria of the Bacillus species secrete two extracellularspecies of protease, a neutral, or metalloprotease, and an alkalineprotease which is functionally a serine endopeptidase, referred to assubtilisin. Secretion of these proteases has been linked to thebacterial growth cycle, with greatest expression of protease during thestationary phase, when sporulation also occurs. Joliffe et al. (1980, J.Bacterial 141:1199-1208) has suggested that Bacillus proteases functionin cell wall turnover.

Subtilisin

A serine protease is an enzyme which catalyzes the hydrolysis of peptidebonds and in which there is an essential serine residue at the activesite (White, Handler and Smith, 1973 “Principles of Biochemistry,” FifthEdition, McGraw-Hill Book Company, N.Y., pp. 271-272).

The bacterial serine proteases have molecular weights in the range of20,000 to 45,000. They are inhibited by diisopropylfluorophosphate, butin contrast to metalloproteases, are resistant to ethylene diaminotetraacetic acid (EDTA) (although they are stabilized at hightemperatures by calcium ions). They hydrolyze simple terminal esters andare similar in activity to eukaryotic chymotrypsin, also a serineprotease. A more narrow term, alkaline protease, covering a sub-group,reflects the high pH optimum of some of the serine proteases, from pH9.0 to 11.0 (for review, see Priest, 1977, Bacteriological Rev.41:711-753).

In relation to the present invention a subtilisin is a serine proteaseproduced by Gram-positive bacteria or fungi. A wide variety ofsubtilisins have been identified and the amino acid sequence of a numberof subtilisins have been determined. These include at least sixsubtilisins from Bacillus strains, namely, subtilisin 168, subtilisinBPN′, subtilisin Carlsberg, subtilisin DY, subtilisin amylosacchariticusand mesentericopeptidase (Kurihara et al., 1972, J. Biol. Chem.247:5629-5631; Wells et al., 1983, Nucleic Acids Res. 11:7911-7925;Stahl and Ferrari, 1984, J. Bacteriol. 159:811-819, Jacobs et al., 1985,Nucl. Acids Res. 13:8913-8926; Nedkov et al., 1985, Biol. Chem.Hoppe-Seyler 366:421-430, Svendsen et al., 1986, FEBS Lett 196:228-232),one subtilisin from an actinomycetales, thermitase fromThermoactinomyces vulgaris (Meloun et al., 1985, FEBS Lett. 1983:195-200) and one fungal subtilisin, proteinase K from Tritirachium album(Jany and Mayer, 1985, Biol. Chem. Hoppe-Seyler 366:584-492).

Subtilisins are well-characterized physically and chemically. Inaddition to knowledge of the primary structure (amino acid sequence) ofthese enzymes, over 50 high resolution X-ray structures of subtilisinhave been determined which delineate the binding of substrate,transition state, products, at least three different protease inhibitorsand define the-structural consequences for natural variation (Kraut,1977, Ann. Rev. Biochem. 46:331-358).

In the context of this invention, a subtilisin variant or mutatedsubtilisin protease means a subtilisin that has been produced by anorganism which is expressing a mutant gene derived from a parentmicroorganism which possessed an original or parent gene and whichproduced a corresponding parent enzyme, the parent gene having beenmutated in order to produce the mutant gene from which said mutatedsubtilisin protease is produced when expressed in a suitable host.

Random and site-directed mutations of the subtilisin gene have botharisen from knowledge of the physical and chemical properties of theenzyme and contributed information relating to subtilisin's catalyticactivity, substrate specificity, tertiary structure, etc. (Wells et al.,1987, Proc. Natl. Acad. Sci. U.S.A. 84; 1219-1223; Wells et al., 1986,Phil. Trans. R. Soc. Lond. A. 317:415-423: Hwang and Warshel, 1987,Biochem. 26:2669-2673; Rao et al., 1987, Nature 328:551-554).

Especially site-directed mutagenesis of the subtilisin genes hasattracted much attention, and various mutations are described in thefollowing patent applications and patents:

EP Publ. No. 130756 (GENENTECH) (corresponding to U.S. Pat. No.4,760,025 (GENENCOR)) relating to site specific or randomly generatedmutations in “carbonyl hydrolases” and subsequent screening of themutated enzymes for various properties, such as k_(cat)/k_(m) ratio,pH-activity profile and oxidation stability. Apart from revealing thatsite-specific mutation is feasible and that mutation of subtilisin BPN′in certain specified positions, i.e. ⁻¹Tyr, ³²Asp, ¹⁵⁵Asn, ¹⁰⁴Tyr,²²²Met, ¹⁶⁶Gly, ⁶⁴His, ¹⁶⁹Gly, ¹⁸⁹Phe, ³³Ser, ²²¹Ser, ²¹⁷Tyr, ¹⁵⁶Glu or¹⁵²Ala, provide for enzymes exhibiting altered properties, thisapplication does not contribute to solving the problem of deciding whereto introduce mutations in order to obtain enzymes with desiredproperties.

EP Publ. No. 214435 (HENKEL) relating to cloning and expression ofsubtilisin Carlsberg and two mutants thereof. In this application noreason for mutation of ¹⁵⁸Asp to ¹⁵⁸Ser and ¹⁶¹Ser to ¹⁶¹Asp isprovided.

In International Patent Publication No. WO 87/04461 (AMGEN) it isproposed to reduce the number of Asn-Gly sequences present in the parentenzyme in order to obtain mutated enzymes exhibiting improved pH andheat stabilities. In the application, emphasis is put on removing,mutating, or modifying the ¹⁰⁹Asn and the ²¹⁸Asn residues in subtilisinBPN′.

International patent publication No. WO 87/05050 (GENEX) disclosesrandom mutation and subsequent screening of a large number of mutants ofsubtilisin BPN′ for improved properties. In the application, mutationsare described in positions ²¹⁸Asn, ¹³¹Gly, ²⁵⁴Thr, ¹⁶⁶Gly, ¹¹⁶Ala,¹⁸⁸Ser, ¹²⁶Leu and ⁵³Ser.

In EP Application No. 87303761 (GENENTECH) it is described how homologyconsiderations at both primary and tertiary structural levels may beapplied to identify equivalent amino acid residues whether conserved ornot. This information together with the inventors' knowledge of thetertiary structure of subtilisin BPN′ led the inventors to select anumber of positions susceptible to mutation with an expectation ofobtaining mutants with altered properties. The positions so identifiedare: ¹²⁴Met, ²²²Met, ¹⁰⁴Tyr, ¹⁵²Ala, ¹⁵⁶Glu, ¹⁶⁶Gly, ¹⁶⁹Gly, ¹⁸⁹Phe,²¹⁷Tyr. Also ¹⁵⁵Asn, ²¹Tyr, ²²Thr, ²⁴Ser, ³²Asp, ³³Ser, ³⁶Asp, ⁴⁶Gly,⁴⁸Ala, ⁴⁹Ser, ⁵⁰Met, ⁷⁷Asn, ⁸⁷Ser, ⁹⁴Lys, ⁹⁵Val, ⁹⁶Leu, ¹⁰⁷Ile, ¹¹⁰Gly,¹⁷⁰Lys, ¹⁷¹Tyr, ¹⁷²Pro, ¹⁹⁷Asp, ¹⁹⁹Met, ²⁰⁴Ser, ²¹³Lys and ²²¹Ser. Thepositions are identified as being expected to influence variousproperties of the enzyme. In addition, a number of mutations areexemplified to support these suggestions. In addition to singlemutations in these positions, the inventors also performed a number ofmultiple mutations. Furthermore, the inventors identified ²¹⁵Gly, ⁶⁷His,¹²⁶Leu, ¹³⁵Leu and amino acid residues within the segments 97-103,126-129, 213-215 and 152-172 as having interest, but mutations in any ofthese positions are not exemplified.

EP Publ. No. 260105 (GENENCOR) describes modification of certainproperties in enzymes containing a catalytic triad by selecting an aminoacid residue within about 15Å from the catalytic triad and replacing theselected amino acid residue with another residue. Enzymes of thesubtilisin type described in the present specification are specificallymentioned as belonging to the class of enzymes containing a catalytictriad. In subtilisins, positions 222 and 217 are indicated as preferredpositions for replacement.

International Patent Publication No. WO 88/06624 (GIST-BROCADES NV)discloses the DNA and amino acid sequences of a subtilisin proteasedesignated PB92 which is almost 100% homologous to the amino acidsequence of Subtilisin 309.

International Patent Publication No. WO 88/07578 (GENENTECH) claimsmutated enzymes derived from a precursor enzyme by replacement ormodification of at least one catalytic group of an amino acid residue.The inventors state that by doing so a mutated enzyme is obtained thatis reactive with substrates containing the modified or replacedcatalytic group (substrate-assisted catalysis).

The general theory is based on B. amyloliquefaciens subtilisin (BPN′),where modifications have been described in positions ⁶⁴His that wasmodified into ⁶⁴Ala alone or in combination with a “helper” mutation ofSer-24-Cys. Modifications are also suggested in the amino acid residues³²Asp and ²²¹Ser and a “helper” mutation of Ala-48-Glu.

International Patent Publication No. WO 88/08028 (GENEX) disclosesgenetic engineering around metal ion binding sites for the stabilizationof proteins. This publication also uses Subtilisin BPN′ as an exampleand points at the following amino acid residues as candidates forsubstitution ¹⁷²Pro (P172D, P172E), ¹³¹Gly (G131D), ⁷⁶Asn (N76D;N76D+P172D(E)), ⁷⁸Ser (S78D). Further, suggestions are made for thecombined mutants N76D+S78D+G131D+P172D(E); N76D+G131D; S78D+G131D;S78D+P172D(E) and S78D+G131D+P172D(E).

International Patent Publication No. WO 88/08033 (AMGEN) discloses anumber of subtilisin analogues having a modified calcium binding siteand either Asn or Gly replaced in any Asn-Gly sequence present in themolecule thereby obtaining enzymes exhibiting improved thermal and pHstability. One of the calcium binding sites is disclosed as involvingthe amino acid residues ⁴¹Asp, ⁷⁵Leu, ⁷⁶Asn, ⁷⁷Asn, ⁷⁸Ser, ⁷⁹Ile,⁸⁰UGly, ⁸¹Val, ²⁰⁸Thr and ²¹⁴Tyr; other potential calcium binding sitesare suggested at ¹⁴⁰Asp and ¹⁷²Pro; ¹⁴Pro and ²⁷¹Gln; and ¹⁷²Pro and¹⁹⁵Glu or ¹⁹⁷Asp. Also mutating the ¹⁰⁹Asn and ²¹⁸Asn positions issuggested. Mutants produced are N109S, N109S+N218S, N76D+N109S+N218S,N76D+N77D+N109S+N218S, N76D+I79E+N109S+N218S.

International Patent Publication No. WO 88/08164 (GENEX) describes amethod for identifying residues in a protein which may be substituted bya cysteine to permit formation of potentially stabilizing disulfidebonds. The method is based on detailed knowledge of the threedimensional structure of the protein and uses a computer for selectingthe positions. In relation to subtilisin proteases, Subtilisin BPN′ wasused as a model system. Using the method on Subtilisin BPN′ resulted inthe suggestion of 11 candidates for introducing disulfide bonds, i.e.1:T22C+S87C, 2:V26C+L235C, 3:G47C+P57C, 4:M50C+N109C, 5:E156C+T164C,6:V165C+K170C, 7:V165C+S191C, 8:Q206C+A216C, 9:A230C+V270C,10:I234C+A274C and 11:H238C+W241C. Of these, four were produced, i.e. 1,2, 4 and 8, of which two did not provide any stabilizing effect, i.e. 2and 4. Further mutants were produced by combining two of these mutantswith each other and one with another mutation, viz. T22C+S87C+N218S andT22C+S87C+Q206C+A216C. Also, a number of further unsuccessful mutantswere produced, viz. AlC+S78C, S24C+S87C, K27C+S89C, A85C+A232C,I122C+V147C, S249C+A273C and T253C+A273C.

In addition, it has been shown by Thomas, Russell and Fersht, Nature318, 375-376 (1985) that the exchange of ⁹⁹Asp into ⁹⁹Ser in subtilisinBPN′ changes the pH dependency of the enzyme.

In a subsequent article J. Mol. Biol. 193, 803-813 (1987), the sameauthors discussed the substitution of ¹⁵⁶Ser for ¹⁵⁶Glu.

Both of these mutations are within a distance of about 15 Å from theactive ⁶⁴His.

In Nature 328, 496-500 (1987) Russel and Fersht discuss the results oftheir experiments and present rules for changing pH-activity profiles bymutating an enzyme to obtain changes in surface charge.

Isoelectric Point (pI_(o))

The isoelectric point, pI_(o), is defined as the pH value where theenzyme molecule complex (with optionally attached metal or other ions)is neutral, i.e. the sum of electrostatic charges. (net electrostaticcharge=NEC) on the complex is equal to zero. In this sum of courseconsideration of the positive or negative nature of the individualelectrostatic charges must be taken into account.

The isoelectric point is conveniently calculated by using equilibriumconsiderations using pK values for the various charged residues in theenzyme in question and then finding by iteration the pH value where theNEC of the enzyme molecule is equal to zero.

One problem with this calculation is that the pK values for the chargedresidues are dependent on their environment and consequently subject tovariation. However, very good results are obtainable by allocatingspecific approximate pK values to the charged residues independently ofthe actual value. It is also possible to perform more sophisticatedcalculations, partly taking the environment into consideration.

The pI_(o) may also be determined experimentally by isoelectric focusingor by titrating a solution containing the enzyme. In addition, thevarious pK values for the charged residues may be determinedexperimentally by titration.

Industrial Applications of Subtilisins

Proteases such as subtilisins have found much utility in industry,particularly in detergent formulations, as they are useful for removingproteinaceous stains.

At present, the following proteases are known, many of which aremarketed in large quantities in many countries of the world:

Subtilisin BPN′ or Novo, available from e.g. SIGMA, St. Louis, U.S.A.;

Subtilisin Carlsberg, marketed by Novo-Nordisk A/S (Denmark) asALCALASE® and by IBIS (Holland) as MAXATASE®;

A Bacillus lentus subtilisin, marketed by NOVO INDUSTRI A/S (Denmark) asSAVINASE®;

Enzymes closely resembling SAVINASE® such as MAXACAL® marketed by IBISand OPTICLEAN® marketed by MILES KALI CHEMIE (FRG);

A Bacillus lentus subtilisin, marketed by Novo Nordisk A/S (Denmark) asESPERASE®; and

KAZUSASE® marketed by SHOWA DENKO (Japan).

However, in order to be effective, these enzymes must not only exhibitactivity under washing conditions, but also be compatible with otherdetergent components during detergent production and storage.

For example, subtilisins may be used in combination with other enzymesactive against other substrates and, therefore, the selected subtilisinshould possess stability towards and preferably should not catalyze thedegradation of the other enzymes. In addition, the chosen subtilisinshould be resistant to the action from other components in the detergentformulation, such as bleaching agents, oxidizing agents, etc., inparticular an enzyme to be used in a detergent formulation should bestable with respect to the oxidizing power, calcium binding propertiesand pH conditions rendered by the non-enzymatic components in thedetergent during storage and in the wash liquor during wash.

The ability of an enzyme to catalyze the degradation of variousnaturally occurring substrates present on the objects to be cleanedduring e.g. wash is often referred to as its washing ability,washability, detergency or wash performance. Throughout this applicationthe term wash performance will be used to encompass this property.

Naturally occurring subtilisins have been found to possess propertieswhich are highly variable in relation to their washing power or abilityunder variations in parameters such as pH. Several of the above marketeddetergent proteases, indeed, have a better performance than thosemarketed about 20 years ago, but for optimal performance each enzyme hasits own specific conditions regarding formulation and wash conditions,e.g. pH, temperature, ionic strength (═I), active system (tensides,surfactants, bleaching agent, etc.), builders, etc.

As a result, it has been found that an enzyme possessing desirableproperties at low pH and low I may be less attractive at more alkalineconditions and high I, or an enzyme exhibiting fine properties at highpH and high I may be less attractive at low pH, low I conditions.

The advent and development of recombinant DNA techniques has had aprofound influence in the field of protein chemistry.

It has been envisaged that these techniques will make it possible todesign peptides and proteins, such as enzymes and hormones according todesired specifications, enabling the production of compounds exhibitingdesired properties.

It is now possible to construct enzymes having desired amino acidsequences, and as indicated above a fair amount of research has beendevoted to designing subtilisins with altered properties. The proposalsinclude the technique of producing and screening a large number ofmutated enzymes as described in EP Publ. No. 130756 (GENENTECH) (U.S.Pat. No. 4,760,025 (GENENCOR)) and International patent Publ. No. WO87/05050 (GENEX). These methods correspond to the classical method ofisolating native enzymes and screening them for their properties, but ismore efficient through the knowledge of the presence of a large numberof different mutant enzymes.

Since a subtilisin enzyme typically comprises 275 amino acid residueseach capable of being 1 out of 20 possible naturally occurring aminoacids, one very serious drawback in that procedure is the very largenumber of mutations generated that has to be submitted to a preliminaryscreening prior to further testing of selected mutants showinginteresting characteristics at the first screening, since no guidance isindicated in determining which amino acid residues to change in order toobtain a desired enzyme with improved properties for the use inquestion, such as, in this case formulating detergent compositionsexhibiting improved wash performance under specified conditions of thewash liquor.

A procedure as outlined in these patent applications will consequentlyonly be slightly better than the traditional random mutation procedureswhich have been known for years.

The other known techniques relate to changing specific properties, suchas transesterification and hydrolysis rate (EP Publ. No. 260105(GENENCOR)), pH-activity profile (Thomas, Russell and Fersht, supra) andsubstrate specificity (International Patent Publ. No. WO 88/07578(GENENTECH)). None of these publications relates to changing the washperformance of enzymes.

A further technique that has evolved is using the detailed informationon the three dimensional structure of proteins for analyzing thepotential consequences of substituting certain amino acids. Thisapproach is described in EP 260105 (GENENCOR), WO 88/07578 (GENENTECH),WO 88/08028 (GENEX), WO 88/08033 (AMGEN) and WO 88/08164 (GENEX).

Thus, as indicated above, no relationship has yet been identifiedbetween well defined properties of an enzyme such as those mentionedabove and the wash performance of an enzyme.

In unpublished International Patent Application No. PCT/DK 88/00002(NOVO INDUSTRI A/S), it is proposed to use the concept of homologycomparison to determine which amino acid positions should be selectedfor mutation and which amino acids should be substituted in thesepositions in order to obtain a desired change in wash performance.

By using such a procedure the task of screening is reduced drastically,since the number of mutants generated is much smaller, but with thatprocedure it is only foreseen that enzymes exhibiting the combineduseful properties of the parent enzyme and the enzyme used in thecomparison may be obtained.

The problem seems to be that although much research has been directed atrevealing the mechanism of enzyme activity, still only little is knownabout the factors in structure and amino acid residue combination thatdetermine the properties of enzymes in relation to their washperformance.

Consequently, there still exists a need for further improvement andtailoring of enzymes to wash systems as well as a better understandingof the mechanism of protease action in the practical use of cleaning ordetergent compositions. Such an understanding could result in ruleswhich may be applied for selecting mutations that with a reasonabledegree of certainty will result in an enzyme exhibiting improved washperformance under specified conditions in a wash liquor.

Lipases in Detergents

Examples of known lipase-containing detergent compositions are providedby EP 0 205 208 and 0 206 390 (Unilever), which relate to lipasesderived from Ps. fluorescens, P gladioli and Chromobacter in detergentcompositions.

EP 0 214 761 (Novo) and EP 0 258 068 (Novo) give a detailed descriptionof lipases from certain microorganisms and disclose the use thereof indetergent additives and detergent compositions. The lipases disclosed inEP 0 214 761 are derived from organisms of the species Pseudomonascepacia. The lipases disclosed in EP 0 258 068 are derived fromorganisms of the genus Thermomyces/Humicola.

A difficulty with the simultaneous incorporation of both lipases andproteases into such compositions is that the protease tends to attachthe lipase.

Measures have been proposed to mitigate this disadvantage.

One such attempt is represented by EP 0 271 154 (Unilever) whereincertain selected proteases with an isoelectric point of less than 10 areshown to combine advantageously with lipases.

Another attempt is described in WO 89/04361 (Novo) which concernsdetergent compositions containing lipase derived from Pseudomonas andprotease derived from Fusarium or protease of a subtilisin type whichhas been mutated in its amino acid sequence at position 166, 169 or 222in certain ways. It was reported that there was some reduction in thedegree of attack upon the lipase by the particular proteases described.

ABBREVIATIONS AMINO ACIDS A = Ala = Alanine V = Val = Valine L = Leu =Leucine I = Ile = Isoleucine P = Pro = Proline F = Phe = Phenylalanine W= Trp = Tryptophan M = Met = Methionine G = Gly = Glycine S = Ser =Serine T = Thr = Threonine C = Cys = Cysteine Y = Tyr = Tyrosine N = Asn= Asparagine Q = Gln = Glutamine D = Asp = Aspartic Acid E = Glu =Glutamic Acid K = Lys = Lysine R = Arg = Arginine H = His = HistidineNUCLEIC ACID BASES A = Adenine G = Guanine C = Cytosine T = Thymine(only in DNA) U = Uracil (only in RNA)

Mutations

In describing the various mutants produced or contemplated according tothe invention, the following nomenclatures were adapted for ease ofreference: Original amino acid(s) position(s) substituted amino acid(s).

Accordingly, the substitution of Glutamic acid for glycine in position195 is designated as:

Gly 195 Glu or G195E.

A deletion of glycine in the same position is designated as:

Gly 195 * or G195*

and an insertion of an additional amino acid residue such as lysine isdesignated as:

Gly 195 GlyLys or G195GK.

Where a deletion is indicated in Table I or present in a subtilisin notindicated in Table I, an insertion in such a position is indicated as:

* 36 Asp or *36D for insertion of an aspartic acid in position 36.

Multiple mutations are separated by pluses, i.e.:

Arg 170 Tyr+Gly 195 Glu or R170Y+G195E representing mutations inpositions 170 and 195 substituting tyrosine and glutamic acid forarginine and glycine, respectively.

TABLE I COMPARISON OF AMINO ACID SEQUENCE FOR VARIOUS PROTEASES a =subtilisin BPN' (Wells et al, 1983, supra) b = subtilisinamylosacchariticus (Kurihara et al, 1972, supra) c = subtilisin 168(Stahl and Ferrari, 1984, supra) d = subtilisin mesentericopeptidase(Svendsen et al, 1986, supra) e = subtilisin DY (Nedkov et al, 1985,supra) f = subtilisin Carlsberg (Smith et al, 1968, supra) g =subtilisin Carlsberg (Jacobs et al, 1985, supra) h = subtilisin 309(International Patent Application No. PCT/DK 88/00002) i = subtilisin147 (International Patent Application No. PCT/DK 88/00002) j =thermitase (Meloun et al, 1985, supra) k = proteinase K (Betzel et al,1988, Eur. J. Biochem. 178:155 ff and Gunkel et al, 1989, Eur. J.Biochem. 179:185 ff) l = agualysin (Kwon et al, 1988, Eur. J. Biochem.173:491 ff) m = Bacillus PB92 protease (European Patent Publication No.0 283 075) n = Protease TW7 (Tritirachium album) (International PatentApplication No. PCT/US88/01040) o = Protease TW3 (Tritirachium album)(International Patent Application No. PCT/US88/01040) * = assigneddeletion

No:               1                   10 a)*-*-*-*-*-*-*-A-Q-S-*-V-P-Y-G-V-S-Q-I-K-*-*-*-*-*-A-P-A- b)*-*-*-*-*-*-*-A-Q-S-*-V-P-Y-G-V-S-Q-I-K-*-*-*-*-*-A-P-A- c)*-*-*-*-*-*-*-A-Q-S-*-V-P-Y-G-V-S-Q-I-K-*-*-*-*-*-A-P-A- d)*-*-*-*-*-*-*-A-Q-S-*-V-P-Y-G-V-S-Q-I-K-*-*-*-*-*-A-P-A- e)*-*-*-*-*-*-*-A-Q-T-*-V-P-Y-G-I-P-L-I-K-*-*-*-*-*-A-D-K- f)*-*-*-*-*-*-*-A-Q-T-*-V-P-Y-G-I-P-L-I-K-*-*-*-*-*-A-D-K- g)*-*-*-*-*-*-*-A-Q-T-*-V-P-Y-G-I-P-L-I-K-*-*-*-*-*-A-D-K- h)*-*-*-*-*-*-*-A-Q-T-*-V-P-W-G-I-S-R-V-Q-*-*-*-*-*-A-P-A- i)*-*-*-*-*-*-*-*-Q-T-*-V-P-W-G-I-S-F-I-N-*-*-*-*-*-T-Q-Q- j)Y-T-P-N-D-P-Y-F-S-S-*-R-Q-Y-G-P-Q-K-I-Q-*-*-*-*-*-A-P-Q- k)*-*-*-*-*-*-A-A-Q-T-N-A-P-W-G-L-A-R-I-S-S-T-S-P-G-T-S-T- l)*-*-*-*-*-*-A-T-Q-S-P-A-P-W-G-L-D-R-I-D-Q-R-D-L-P-L-S-N- m)*-*-*-*-*-*-*-A-Q-S-*-V-P-W-G-I-S-R-V-Q-*-*-*-*-*-A-P-A- n)*-*-*-*-*-*-A-T-Q-E-D-A-P-W-G-L-A-R-I-S-S-Q-E-P-G-G-T-T- o)*-*-*-*-*-*-A-E-Q-R-N-A-P-W-G-L-A-R-I-S-S-T-S-P-G-T-S-T- No:        20                  30                  40 a)L-H-S-Q-G-Y-T-G-S-N-V-K-V-A-V-I-D-S-G-I-D-S-S-H-P-D-L-*- b)L-H-S-Q-G-Y-T-G-S-N-V-K-V-A-V-I-D-S-G-I-D-S-S-H-P-D-L-*- c)L-H-S-Q-G-Y-T-G-S-N-V-K-V-A-V-I-D-S-G-I-D-S-S-H-P-D-L-*- d)L-H-S-Q-G-Y-T-G-S-N-V-K-V-A-V-I-D-S-G-I-D-S-S-H-P-D-L-*- e)V-Q-A-Q-G-Y-K-G-A-N-V-K-V-G-I-I-D-T-G-I-A-A-S-H-T-D-L-*- f)V-Q-A-Q-G-F-K-G-A-N-V-K-V-A-V-L-D-T-G-I-Q-A-S-H-P-D-L-*- g)V-Q-A-Q-G-F-K-G-A-N-V-K-V-A-V-L-D-T-G-I-Q-A-S-H-P-D-L-*- h)A-H-N-R-G-L-T-G-S-G-V-K-V-A-V-L-D-T-G-I-*-S-T-H-P-D-L-*- i)A-H-N-R-G-I-F-G-N-G-A-R-V-A-V-L-D-T-G-I-*-A-S-H-P-D-L-*- j)A-W-*-D-I-A-E-G-S-G-A-K-I-A-I-V-D-T-G-V-Q-S-N-H-P-D-L-A- k)Y-Y-Y-D-E-S-A-G-Q-G-S-C-V-Y-V-I-D-T-G-I-E-A-S-H-P-E-F-*- l)S-Y-T-Y-T-A-T-G-R-G-V-N-V-Y-V-I-D-T-G-I-R-T-T-H-R-E-F-*- m)A-H-N-R-G-L-T-G-S-G-V-K-V-A-V-L-D-T-G-I-*-S-T-H-P-D-L-*- n)Y-R-Y-D-D-S-A-G-T-G-T-C-A-Y-I-I-D-T-G-I-Y-T-N-H-T-D-F-*- o)Y-R-Y-D-D-S-A-G-Q-G-T-C-V-Y-V-I-D-T-G-V-E-A-S-H-P-E-F-*- No:                50                      60 a)*-K-V-A-G-G-A-S-M-V-P-S-E-T-N-P-F-*-*-Q-D-N-N-S-H-G-T-H-V- b)*-N-V-R-G-G-A-S-F-V-P-S-E-T-N-P-Y-*-*-Q-D-G-S-S-H-G-T-H-V- c)*-N-V-R-G-G-A-S-F-V-P-S-E-T-N-P-Y-*-*-Q-D-G-S-S-H-G-T-H-V- d)*-N-V-R-G-G-A-S-F-V-P-S-E-T-N-P-Y-*-*-Q-D-G-S-S-H-G-T-H-V- e)*-K-V-V-G-G-A-S-F-V-S-G-E-S-*-Y-N-*-*-T-D-G-N-G-H-G-T-H-V- f)*-N-V-V-G-G-A-S-F-V-A-G-E-A-*-Y-N-*-*-T-D-G-N-G-H-G-T-H-V- g)*-N-V-V-G-G-A-S-F-V-A-G-E-A-*-Y-N-*-*-T-D-G-N-G-H-G-T-H-V- h)*-N-I-R-G-G-A-S-F-V-P-G-E-P-*-S-T-*-*-Q-D-G-N-G-H-G-T-H-V- i)*-R-I-A-G-G-A-S-F-I-S-S-E-P-*-S-Y-*-*-H-D-N-N-G-H-G-T-H-V- j)G-K-V-V-G-G-W-D-F-V-D-N-D-S-T-P-*-*-*-Q-N-G-N-G-H-G-T-H-C- k)*-*-*-E-G-R-A-Q-M-V-K-T-Y-Y-Y-S-S-*-*-R-D-G-N-G-H-G-T-H-C- l)*-*-*-G-G-R-A-R-V-G-Y-D-A-L-G-G-N-G-*-Q-D-C-N-G-H-G-T-H-V- m)*-N-I-R-G-G-A-S-F-V-P-G-E-P-*-S-T-*-*-Q-D-G-N-G-H-G-T-H-V- n)*-*-*-G-G-R-A-K-F-L-K-N-F-A-G-D-G-Q-D-T-D-G-N-G-H-G-T-H-V- o)*-*-*-E-G-R-A-Q-M-V-K-T-Y-Y-A-S-S-*-*-R-D-G-N-G-H-G-T-H-C- No:  70                    80                  90 a)A-G-T-V-A-A-L-*-N-N-S-I-G-V-L-G-V-A-P-S-A-S-L-Y-A-V-K-V- b)A-G-T-I-A-A-L-*-N-N-S-I-G-V-L-G-V-A-P-S-A-S-L-Y-A-V-K-V- c)A-G-T-I-A-A-L-*-N-N-S-I-G-V-L-G-V-S-P-S-A-S-L-Y-A-V-K-V- d)A-G-T-I-A-A-L-*-N-N-S-I-G-V-L-G-V-A-P-S-A-S-L-Y-A-V-K-V- e)A-G-T-V-A-A-L-*-D-N-T-T-G-V-L-G-V-A-P-N-V-S-L-Y-A-I-K-V- f)A-G-T-V-A-A-L-*-D-N-T-T-G-V-L-G-V-A-P-S-V-S-L-Y-A-V-K-V- g)A-G-T-V-A-A-L-*-D-N-T-T-G-V-L-G-V-A-P-S-V-S-L-Y-A-V-K-V- h)A-G-T-I-A-A-L-*-N-N-S-I-G-V-L-G-V-A-P-S-A-E-L-Y-A-V-K-V- i)A-G-T-I-A-A-L-*-N-N-S-I-G-V-L-G-V-A-P-S-A-D-L-Y-A-V-K-V- j)A-G-I-A-A-A-V-T-N-N-S-T-G-I-A-G-T-A-P-K-A-S-I-L-A-V-R-V- k)A-G-T-V-G-S-*-R-*-*-*-*-*-T-Y-G-V-A-K-K-T-Q-L-F-G-V-K-V- l)A-G-T-I-G-G-V-*-*-*-*-*-*-T-Y-G-V-A-K-A-V-N-L-Y-A-V-R-V- m)A-G-T-I-A-A-L-*-N-N-S-I-G-V-L-G-V-A-P-N-A-E-L-Y-A-V-K-V- n)A-G-T-V-G-G-T-*-*-*-*-*-*-T-Y-G-V-A-K-K-T-S-L-F-A-V-K-V- o)A-G-T-I-G-S-*-R-*-*-*-*-*-T-Y-G-V-A-K-K-T-Q-I-F-G-V-K-V- No:        100                 110                     120 a)L-G-A-D-G-S-G-Q-Y-S-W-I-I-N-G-I-E-W-*-A-I-A-*-N-N-M-D-*- b)L-D-S-T-G-S-G-Q-Y-S-W-I-I-N-G-I-E-W-*-A-I-A-*-N-N-M-D-*- c)L-D-S-T-G-S-G-Q-Y-S-W-I-I-N-G-I-E-W-*-A-I-A-*-N-N-M-D-*- d)L-D-S-T-G-S-G-Q-Y-S-W-I-I-N-G-I-E-W-*-A-I-A-*-N-N-M-D-*- e)L-N-S-S-G-S-G-T-Y-S-A-I-V-S-G-I-E-W-*-A-T-Q-*-N-G-L-D-*- f)L-N-S-S-G-S-G-S-Y-S-G-I-V-S-G-I-E-W-*-A-T-T-*-N-G-M-D-*- g)L-N-S-S-G-S-G-T-Y-S-G-I-V-S-G-I-E-W-*-A-T-T-*-N-G-M-D-*- h)L-G-A-S-G-S-G-S-V-S-S-I-A-Q-G-L-E-W-*-A-G-N-*-N-G-M-H-*- i)L-D-R-N-G-S-G-S-L-A-S-V-A-Q-G-I-E-W-*-A-I-N-*-N-N-M-H-*- j)L-D-N-S-G-S-G-T-W-T-A-V-A-N-G-I-T-Y-*-A-A-D-*-Q-G-A-K-*- k)L-D-D-N-G-S-G-Q-Y-S-T-I-I-A-G-M-D-F-V-A-S-D-K-N-N-R-N-C- l)L-D-C-N-G-S-G-S-T-S-G-V-I-A-G-V-D-W-V-*-T-*-R-N-H-R-R-P- m)L-G-A-S-G-S-G-S-V-S-S-I-A-Q-G-L-E-W-*-A-G-N-*-N-G-M-H-*- n)L-D-A-N-G-Q-G-S-N-S-G-V-I-A-G-M-D-F-V-T-K-D-A-S-S-Q-N-C- o)L-N-D-Q-G-S-G-Q-Y-S-T-I-I-S-G-M-D-F-V-A-N-D-Y-R-N-R-N-C- No:                              130             140 a)*-*-*-*-V-I-N-M-S-L-G-G-P-S-G-S-A-A-L-K-A-A-V-D-K-A-V-A- b)*-*-*-*-V-I-N-M-S-L-G-G-P-S-G-S-T-A-L-K-T-V-V-D-K-A-V-S- c)*-*-*-*-V-I-N-M-S-L-G-G-P-T-G-S-T-A-L-K-T-V-V-D-K-A-V-S- d)*-*-*-*-V-I-N-M-S-L-G-G-P-T-G-S-T-A-L-K-T-V-V-D-K-A-V-S- e)*-*-*-*-V-I-N-M-S-L-G-G-P-S-G-S-T-A-L-K-Q-A-V-D-K-A-Y-A- f)*-*-*-*-V-I-N-M-S-L-G-G-A-S-G-S-T-A-M-K-Q-A-V-D-N-A-Y-A- g)*-*-*-*-V-I-N-M-S-L-G-G-P-S-G-S-T-A-M-K-Q-A-V-D-N-A-Y-A- h)*-*-*-*-V-A-N-L-S-L-G-S-P-S-P-S-A-T-L-E-Q-A-V-N-S-A-T-S- i)*-*-*-*-I-I-N-M-S-L-G-S-T-S-G-S-S-T-L-E-L-A-V-N-R-A-N-N- j)*-*-*-*-V-I-S-L-S-L-G-G-T-V-G-N-S-G-L-Q-Q-A-V-N-Y-A-W-N- k)P-K-G-V-V-A-S-L-S-L-G-G-G-Y-S-S-S-V-N-S-A-A-A-*-R-L-Q-S- l)A-*-*-*-V-A-N-M-S-L-G-G-G-V-*-S-T-A-L-D-N-A-V-K-N-S-I-A- m)*-*-*-*-V-A-N-L-S-L-G-S-P-S-P-S-A-T-L-E-Q-A-V-N-S-A-T-S- n)P-K-G-V-V-V-N-M-S-L-G-G-P-S-S-S-A-V-N-R-A-A-A-*-E-I-T-S- o)P-N-G-V-V-A-S-M-S-I-G-G-G-Y-S-S-S-V-N-S-A-A-A-*-N-L-Q-Q- No:          150                 160                 170 a)S-G-V-V-V-V-A-A-A-G-N-E-G-T-S-G-S-S-S-T-V-G-Y-P-G-K-Y-P- b)S-G-I-V-V-A-A-A-A-G-N-E-G-S-S-G-S-S-S-T-V-G-Y-P-A-K-Y-P- c)S-G-I-V-V-A-A-A-A-G-N-E-G-S-S-G-S-T-S-T-V-G-Y-P-A-K-Y-P- d)S-G-I-V-V-A-A-A-A-G-N-E-G-S-S-G-S-T-S-T-V-G-Y-P-A-K-Y-P- e)S-G-I-V-V-V-A-A-A-G-N-S-G-S-S-G-S-Q-N-T-I-G-Y-P-A-K-Y-D- f)R-G-V-V-V-V-A-A-A-G-N-S-G-N-S-G-S-T-N-T-I-G-Y-P-A-K-Y-D- g)R-G-V-V-V-V-A-A-A-G-N-S-G-S-S-G-N-T-N-T-I-G-Y-P-A-K-Y-D- h)R-G-V-L-V-V-A-A-S-G-N-S-G-A-*-G-S-I-S-*-*-*-Y-P-A-R-Y-A- i)A-G-I-L-L-V-G-A-A-G-N-T-G-R-*-Q-G-V-N-*-*-*-Y-P-A-R-Y-S- j)K-G-S-V-V-V-A-A-A-G-N-A-G-N-T-A-P-N-*-*-*-*-Y-P-A-Y-Y-S- k)S-G-V-M-V-A-V-A-A-G-N-N-N-A-D-A-R-N-Y-S-*-*-*-P-A-S-E-P- l)A-G-V-V-Y-A-V-A-A-G-N-D-N-A-N-A-C-N-Y-S-*-*-*-P-A-R-V-A- m)R-G-V-L-V-V-A-A-S-G-N-S-G-A-*-G-S-I-S-*-*-*-Y-P-A-R-Y-A- n)A-G-L-F-L-A-V-A-A-G-N-E-A-T-D-A-S-S-S-S-*-*-*-P-A-S-E-E- o)S-G-V-M-V-A-V-A-A-G-N-N-N-A-D-A-R-N-Y-S-*-*-*-P-A-S-E-S- No:             180                 190                 200 a)S-V-I-A-V-G-A-V-D-S-S-N-Q-R-A-S-F-S-S-V-G-P-E-L-D-V-M-A- b)S-T-I-A-V-G-A-V-N-S-S-N-Q-R-A-S-F-S-S-A-G-S-E-L-D-V-M-A- c)S-T-I-A-V-G-A-V-N-S-S-N-Q-R-A-S-F-S-S-A-G-S-E-L-D-V-M-A- d)S-T-I-A-V-G-A-V-N-S-A-N-Q-R-A-S-F-S-S-A-G-S-E-L-D-V-M-A- e)S-V-I-A-V-G-A-V-D-S-N-K-N-R-A-S-F-S-S-V-G-A-E-L-E-V-M-A- f)S-V-I-A-V-G-A-V-D-S-N-S-N-R-A-S-F-S-S-V-G-A-E-L-E-V-M-A- g)S-V-I-A-V-G-A-V-D-S-N-S-N-R-A-S-F-S-S-V-G-A-E-L-E-V-M-A- h)N-A-M-A-V-G-A-T-D-Q-N-N-N-R-A-S-F-S-Q-Y-G-A-G-L-D-I-V-A- i)G-V-M-A-V-A-A-V-D-Q-N-G-Q-R-A-S-F-S-T-Y-G-P-E-I-E-I-S-A- j)N-A-I-A-V-A-S-T-D-Q-N-D-N-K-S-S-F-S-T-Y-G-S-V-V-D-V-A-A- k)S-V-C-T-V-G-A-S-D-R-Y-D-R-R-S-S-F-S-N-Y-G-S-V-L-D-I-F-G- l)E-A-L-T-V-G-A-T-T-S-S-D-A-R-A-S-F-S-N-Y-G-S-C-V-D-L-F-A- m)N-A-M-A-V-G-A-T-D-Q-N-N-N-R-A-S-F-S-Q-Y-G-A-G-L-D-I-V-A- n)S-A-C-T-V-G-A-T-D-K-T-D-T-L-A-E-Y-S-N-F-G-S-V-V-D-L-L-A- o)S-I-C-T-V-G-A-T-D-R-Y-D-R-R-S-S-F-S-N-Y-G-S-V-L-D-I-F-A- No:                  210                     220 a)P-G-V-S-I-Q-S-T-L-P-G-N-*-K-*-Y-G-A-Y-N-G-T-S-M-A-S-P-H- b)P-G-V-S-I-Q-S-T-L-P-G-G-*-T-*-Y-G-A-Y-N-G-T-S-M-A-T-P-H- c)P-G-V-S-I-Q-S-T-L-P-G-G-*-T-*-Y-G-A-Y-N-G-T-S-M-A-T-P-H- d)P-G-V-S-I-Q-S-T-L-P-G-G-*-T-*-Y-G-A-Y-N-G-T-S-M-A-T-P-H- e)P-G-V-S-V-Y-S-T-Y-P-S-N-*-T-*-Y-T-S-L-N-G-T-S-M-A-S-P-H- f)P-G-A-G-V-Y-S-T-Y-P-T-N-*-T-*-Y-A-T-L-N-G-T-S-M-A-S-P-H- g)P-G-A-G-V-Y-S-T-Y-P-T-S-*-T-*-Y-A-T-L-N-G-T-S-M-A-S-P-H- h)P-G-V-N-V-Q-S-T-Y-P-G-S-*-T-*-Y-A-S-L-N-G-T-S-M-A-T-P-H- i)P-G-V-N-V-N-S-T-Y-T-G-N-*-R-*-Y-V-S-L-S-G-T-S-M-A-T-P-H- j)P-G-S-W-I-Y-S-T-Y-P-T-S-*-T-*-Y-A-S-L-S-G-T-S-M-A-T-P-H- k)P-G-T-S-I-L-S-T-W-I-G-G-*-S-*-T-R-S-I-S-G-T-S-M-A-T-P-H- l)P-G-A-S-I-P-S-A-W-Y-T-S-D-T-A-T-Q-T-L-N-G-T-S-M-A-T-P-H- m)P-G-V-N-V-Q-S-T-Y-P-G-S-*-T-*-Y-A-S-L-N-G-T-S-M-A-T-P-H- n)P-G-T-D-I-K-S-T-W-N-D-G-R-T-K-I-I-S-*-*-G-T-S-M-A-S-P-H- o)P-G-T-D-I-L-S-T-W-I-G-G-S-T-R-S-I-S-*-*-G-T-S-M-A-T-P-H- No:      230                 240                 250 a)V-A-G-A-A-A-L-I-L-S-K-H-P-N-W-T-N-T-Q-V-R-S-S-L-E-N-T-T- b)V-A-G-A-A-A-L-I-L-S-K-H-P-T-W-T-N-A-Q-V-R-D-R-L-E-S-T-A- c)V-A-G-A-A-A-L-I-L-S-K-H-P-T-W-T-N-A-Q-V-R-D-R-L-E-S-T-A- d)V-A-G-A-A-A-L-I-L-S-K-H-P-T-W-T-N-A-Q-V-R-D-R-L-E-S-T-A- e)V-A-G-A-A-A-L-I-L-S-K-Y-P-T-L-S-A-S-Q-V-R-N-R-L-S-S-T-A- f)V-A-G-A-A-A-L-I-L-S-K-H-P-N-L-S-A-S-Q-V-R-N-R-L-S-S-T-A- g)V-A-G-A-A-A-L-I-L-S-K-H-P-N-L-S-A-S-Q-V-R-N-R-L-S-S-T-A- h)V-A-G-A-A-A-L-V-K-Q-K-N-P-S-W-S-N-V-Q-I-R-N-H-L-K-N-T-A- i)V-A-G-V-A-A-L-V-K-S-R-Y-P-S-Y-T-N-N-Q-I-R-Q-R-I-N-Q-T-A- j)V-A-G-V-A-G-L-L-A-S-Q-G-R-S-*-*-A-S-N-I-R-A-A-I-E-N-T-A- k)V-A-G-L-A-A-Y-L-M-T-L-G-K-T-T-A-A-S-A-C-R-*-Y-I-A-D-T-A- l)V-A-G-V-A-A-L-Y-L-E-Q-N-P-S-A-T-P-A-S-V-A-S-A-I-L-N-G-A- m)V-A-G-A-A-A-L-V-K-Q-K-N-P-S-W-S-N-V-Q-I-R-N-H-L-K-N-T-A- n)V-A-G-L-G-A-Y-F-L-G-L-G-Q-K-V-Q-G-L-*-C-D-*-Y-M-V-E-K-G- o)V-A-G-L-A-A-Y-L-M-T-L-G-R-A-T-A-S-N-A-C-R-*-Y-I-A-Q-T-A- No:          260                   270       275 a)T-K-L-G-D-S-F-Y-Y-*-G-K-G-L-I-N-V-Q-A-A-A-Q b)T-Y-L-G-D-S-F-Y-Y-*-G-K-G-L-I-N-V-Q-A-A-A-Q c)T-Y-L-G-N-S-F-Y-Y-*-G-K-G-L-I-N-V-Q-A-A-A-Q d)T-Y-L-G-S-S-F-Y-Y-*-G-K-G-L-I-N-V-Q-A-A-A-Q e)T-N-L-G-D-S-F-Y-Y-*-G-K-G-L-I-N-V-E-A-A-A-Q f)T-Y-L-G-S-S-F-Y-Y-*-G-K-G-L-I-N-V-E-A-A-A-Q g)T-Y-L-G-S-S-F-Y-Y-*-G-K-G-L-I-N-V-E-A-A-A-Q h)T-S-L-G-S-T-N-L-Y-*-G-S-G-L-V-N-A-E-A-A-T-R i)T-Y-L-G-S-P-S-L-Y-*-G-N-G-L-V-H-A-G-R-A-T-Q j)D-K-I-S-G-T-G-T-Y-W-A-K-G-R-V-N-A-Y-K-A-V-Q-Y k)N-K-G-D-L-S-N-I-P-F-G-T-V-N-L-L-A-Y-N-N-Y-Q-A l)T-T-G-R-L-S-G-I-G-S-G-S-P-N-R-L-L-Y-S-L-L-S-S-G-S-G m)T-S-L-G-S-T-N-L-Y-*-G-S-G-L-V-N-A-E-A-A-T-R n)L-K-D-V-I-Q-S-V-P-S-D-T-A-N-V-L-I-N-N-G-E-G-S-A

SUMMARY OF THE INVENTION

Further investigations into these problems have now surprisingly shownthat one of the critical factors in the use of subtilisin enzymes indetergent compositions is the adsorption of the enzyme to the substrate,i.e. the material to be removed from textiles, hard surfaces or othermaterials to be cleaned.

Consequently, the present invention relates to mutations of thesubtilisin gene resulting in changed properties of the mutant subtilisinenzyme expressed by such a mutated gene, whereby said mutant subtilisinenzyme exhibits improved behavior in detergent compositions. Mutationsare generated at specific nucleic acids in the parent subtilisin generesponsible for the expression of specific amino acids in specificpositions in the subtilisin enzyme.

The present invention also relates to methods of selecting the positionsand amino acids to be mutated and thereby mutatis mutandis the nucleicacids to be changed in the subtilisin gene in question.

The invention relates, in part, but is not limited to, mutations of thesubtilisin 309 and subtilisin Carlsberg genes and ensuing mutantsubtilisin 309 and Carlsberg enzymes, which exhibit improved washperformance in different detergent compositions resulting in washliquors of varying pH values.

Furthermore, the invention relates to the use of the mutant enzymes incleaning compositions and cleaning compositions comprising the mutantenzymes, especially detergent compositions comprising the mutantsubtilisin enzymes.

It has surprisingly been found that a decrease in the isoelectric pointand hence the net charge of a subtilisin-type protease under washingconditions, can result in not only an improved wash performance of theenzyme but also an improved compatibility with lipase.

It has also been surprisingly found that compatibility of protease withlipase is influenced not only by the pIo but by the positions at whichthe charges are located relative to the active site of the protease: theintroduction of a negative charge or removal of a positive charge closerto the active site gives stronger improvement of compatibility ofprotease with lipase.

Accordingly, the invention provides in one aspect an enzymatic detergentcomposition comprising a lipase and a mutated subtilisin protease,wherein the net molecular electrostatic charge of the mutated proteasehas been changed by insertion, deletion or substitution of amino acidresidues in comparison to the parent protease, and wherein, in saidprotease, there are, relative to said parent protease, fewerpositively-charged amino acid residue(s) and/or more negatively-chargedamino acid residue(s), whereby said subtilisin protease has anisoelectric pH lower than that of said parent protease.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is described further in detail in the following parts ofthis specification with reference to the drawings wherein:

FIG. 1 shows the construction of plasmid pSX88;

FIG. 2 shows a restriction map of plasmid pSX88;

FIG. 3 exemplifies the construction of the mutant subtilisin 309 genesfor expressing the enzymes of the invention;

FIG. 4 shows the restriction map for plasmid pSX92; and

FIG. 5 graphically demonstrates the relationship between pH of maximumperformance and calculated pI of the mutant enzymes of the invention.

DETAILED DESCRIPTION OF THE INVENTION

As stated above, the present invention relates to mutated subtilisins inwhich the amino acid sequence has been changed through mutating the geneof the subtilisin enzyme, which it is desired to modify, in codonsresponsible for the expression of amino acids located on or close to thesurface of the resulting enzyme.

In the context of this invention, a subtilisin is defined as a serineprotease produced by gram-positive bacteria or fungi. In a more narrowsense, applicable to many embodiments of the invention, subtilisin alsomeans a serine protease of gram-positive bacteria. According to anotherdefinition, a subtilisin is a serine protease, wherein the relativeorder of the amino acid residues in the catalytic triad is Asp—His—Ser(positions 32, 64 and 221). In a still more specific sense, many of theembodiments of the invention relate to serine proteases of gram-positivebacteria which can be brought into substantially unambiguous homology intheir primary structure, with the subtilisins listed in Table I above.

Using the numbering system originating from the amino acid sequence ofsubtilisin BPN′ provided in Table I above aligned with the amino acidsequence of a number of other known subtilisins, it is possible toindicate the position of an amino acid residue in a subtilisin enzymeunambiguously. Positions prior to amino acid residue number 1 insubtilisin BPN′ are assigned a negative number, such as −6 for theN-terminal Y in thermitase, or 0 for the N-terminal A in proteinase K.Amino acid residues which are insertions in relation to subtilisin BPN′are numbered by the addition of letters in alphabetical order to thepreceding subtilisin BPN′ number, such as 12a, 12b, 12c, 12d, 12e forthe “insert” S-T-S-P-G in proteinase K between ¹²Ser and ¹³Thr.

Using the above numbering system the positions of interest are:

1, 2, 3, 4, 6, 9, 10, 12, 14, 15, 17, 18, 19, 20, 21, 22, 24, 25, 27,36, 37, 38, 40, 41, 43, 44, 45, 46, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 75, 76, 77, 78, 79, 87, 89, 91, 94, 97, 98, 99, 100,101, 103, 104, 105, 106, 107, 108, 109, 112, 113, 115, 116, 117, 118,120, 126, 128, 129, 130, 131, 133, 134, 136, 137, 140, 141, 143, 144,145, 146, 155, 156, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,170, 171, 172, 173, 181, 182, 183, 184, 185, 186, 188, 189, 191, 192,194, 195, 197, 204, 206, 209, 210, 211, 212, 213,. 214, 215, 216, 217,218, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 247, 248,249, 251, 252, 253, 254, 255, 256, 257, 259, 260, 261, 262, 263, 265,269, 271, 272 and 275.

Isoelectric Point

Assuming that the substrate under washing conditions has anelectrostatic charge opposite to that of the enzyme, it might beexpected that the adsorption and thus the wash performance of the enzymeto the substrate would be improved by increasing the net electrostaticcharge of the enzyme.

However, it was surprisingly found that a decrease in the NEC of theenzyme under such circumstances could result in an improved washperformance of the enzyme.

Stated differently, it was found that changing the isoelectric point ofthe enzyme in a direction to approach a lower pH, also shifted the pH ofoptimum wash performance of the enzyme to a lower value, meaning that inorder to design an enzyme to a wash liquor of low pH, in which theenzyme is to be active, improvement in the wash performance of a knownsubtilisin enzyme may be obtained by mutating the gene for the knownsubtilisin enzyme to obtain a mutant enzyme having a lower pI_(o).

This finding led to experiments showing that the opposite also isfeasible. Meaning that a known subtilisin enzyme may also be designedfor use in high pH detergents by shifting its pI_(o) to higher values,thereby shifting the wash performance pH optimum for the enzyme tohigher pH values.

The present invention therefore in one aspect relates to mutatedsubtilisin proteases, wherein the net electrostatic charge has beenchanged in comparison to the parent protease at the same pH, and whichproteases have, relative to said parent protease, either fewer or morepositively-charged amino acid residue(s) and/or more or fewernegatively-charged amino acid residue(s), or more or fewerpositively-charged amino acid residue(s) and/or fewer or morenegatively-charged amino acid residue(s) among the amino acid residuesat any one or more of the following positions:

1, 2, 3, 4, 6, 9, 10, 12, 14, 15, 17, 18, 19, 20, 21, 22, 24, 25, 27,36, 37, 38, 40, 41, 43, 44, 45, 46, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 75, 76, 77, 78, 79,. 87, 89, 91, 94, 97, 98, 99,100, 101, 103, 104, 105, 106, 107, 108, 109, 112, 113, 115, 116, 117,118, 120, 126, 128, 129, 130, 131, 133, 134, 136, 137, 140, 141, 143,144, 145, 146, 155, 156, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 170, 171, 172, 173, 181, 182, 183, 184, 185, 186, 188, 189, 191,192, 194, 195, 197, 204, 206, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 247,248, 249, 251, 252, 253, 254, 255, 256, 257, 259, 260, 261, 262, 263,265, 269, 271, 272 and 275

and whereby the isoelectric point of said subtilisin protease is loweror higher, respectively, than that of said parent protease.

In a preferred embodiment, the mutant subtilisin proteases of thepresent invention have, relative to said parent protease, either feweror more positively-charged amino acid residue(s) and/or more or fewernegatively-charged amino acid residue(s), or either more or fewerpositively-charged amino acid residue(s) and/or fewer or morenegatively-charged amino acid residue(s), among the amino acid residuesat any one or more of the following positions:

1, 2, 3, 4, 14, 15, 17, 18, 20, 27, 40, 41, 43, 44, 45, 46, 51, 52, 60,61, 62, 75, 76, 78, 79, 91, 94, 97, 100, 105, 106, 108, 112, 113, 117,118, 129, 130, 133, 134, 136, 137, 141, 143, 144, 145, 146, 165, 173,181, 183, 184, 185, 191, 192, 206, 209, 210, 211, 212, 216, 239, 240,242, 243, 244, 245, 247, 248, 249, 251, 252, 253, 255, 256, 257, 259,263, 269, 271 and 272.

In another preferred embodiment, the mutant subtilisin proteases of thepresent invention have, relative to said parent protease, either feweror more positively-charged amino acid residue(s) and/or more or fewernegatively-charged amino acid residue(s), or either more or fewerpositively-charged amino acid residue(s) and/or fewer or morenegatively-charged amino acid residue(s), among the amino acid residuesat any one or more of positions:

1, 2, 3, 4, 14, 15, 17, 18, 20, 27, 40, 41, 43, 44, 45, 46, 51, 52, 60,61, 62, 75, 76, 78, 79, 91, 94, 97, 100, 105, 106, 108, 112, 113, 117,118, 129, 130, 133, 134, 136, 137, 141, 143, 144, 145, 146, 165, 173,181, 183, 184, 185, 191, 192, 206, 209, 210, 211, 212, 216, 239, 240,242, 243, 244, 245, 247, 248, 249, 251, 252, 253, 255, 256, 257, 259,263, 269, 271 and 272

and at least one further mutation affecting an amino acid residueoccupying one of the following positions:

1, 2, 3, 4, 6, 9, 10, 12, 14, 15, 17, 18, 19, 20, 21, 22, 24, 25, 27,36, 37, 38, 40, 41, 43, 44, 45, 46, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 75, 76, 77, 78, 79, 87, 89, 91, 94, 97, 98, 99, 100,101, 103, 104, 105, 106, 107, 108, 109, 112, 113, 115, 116, 117, 118,120, 126, 128, 129, 130, 131, 133, 134, 136, 137, 140, 141, 143, 144,145, 146, 155, 156, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,170, 171, 172, 173, 181, 182, 183, 184, 185, 186, 188, 189, 191, 192,194, 195, 197, 204, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217,218, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 247, 248,249, 251, 252, 253, 254, 255, 256, 257, 259, 260, 261, 262, 263, 265,269, 271, 272 and 275.

In these aspects, the invention in short relates to mutant proteases inwhich the pI_(o) of the mutant protease is lower than the pI_(o) of theparent protease and the pH for optimum wash performance is also lowerthan the pH optimum for the parent protease; or mutant proteases whereinthe pI_(o) of the mutant protease is higher than the pI_(o) of theparent protease and the pH for optimum wash performance is also higherthan the pH optimum for the parent protease.

It is generally believed (Thomas, Russell and Fersht, supra) thatkinetic properties can be influenced by changes in the electrostaticsurface charge in the vicinity of the active site in the enzyme, but ithas now surprisingly been found that changes in the kinetic propertiesof an enzyme can also be brought about by modifying surface chargesremote from the active site.

Consequently, the invention is also considered to embrace mutantsubtilisin enzymes, wherein one or more amino acid residues in adistance of more than 15 Å from the catalytic triad of said enzyme hasbeen changed in comparison to the amino acid sequence of its parentenzyme, in a way to provide for a mutant protease having an isoelectricpoint shifted to achieve the pH for optimum wash performance of theenzyme, which pH optimum should be as close as possible to the pH of thewash liquor, wherein said mutant protease is intended for use.

In certain embodiments of the detergent compositions, the mutatedsubtilisin protease contains, relative to the corresponding parentprotease, an amino acid residue within about 20 Å of the active sitewhich has been changed by substitution, deletion, or adjacent insertion,e.g. at one or more of the following positions:

6, 27, 36-38, 44, 45, 49, 50-59, 61, 62, 89, 91, 98-101, 103-109, 112-3,126-131, 136, 155, 156, 158-160, 162-164, 166, 167, 170, 171, 181, 182,186, 188, 189, 195, 197, 204, 206, 209 and 211-218.

In certain further embodiments of the detergent compositions, themutated subtilisin protease contains, relative to the correspondingparent protease, an amino acid residue at one or more the followingpositions which has been changed by substitution, insertion or deletion:

6, 9, 11, 12, 19, 25, 36, 37, 38, 53, 54, 55, 56, 57, 58, 59, 67, 71,89, 111, 115, 120, 121, 122, 124, 128, 131, 140, 153, 154, 156, 158,159, 160, 161, 162, 163, 164, 165, 166, 168, 170, 172, 175, 180, 182,186, 187, 191, 194, 195, 199, 218, 219, 226, 234, 235, 236, 237, 238,241, 260, 261, 262, 265, 268 and 275.

In further embodiments, the protease mutation can consist of aninsertion of one or more amino acid residues at any of positions 36, 56,159 and 164-166.

For example, the mutant proteases may contain an insertion mutation atposition 36, e.g. the insertion of a negatively-charged amino acidresidue (e.g. D or E), a neutral polar residue (e.g. A, Q or N) or apositive amino acid residue (e.g. R or K).

This is particularly applicable for example to subtilisin proteases 309and 147 and PB92 and any other sequence for which the homology indicatesthat it naturally has an amino acid residue missing at position 36relative to the sequence of subtilisin BPN′.

Insertion mutants at position 36 can have further mutations, for exampleat one or more of positions 120, 170, 195, 235, 251 and/or 76.

Suitable mutations at position 76 are e.g. negatively charged residuessuch as N76D or N76E.

Mutations at position 36 (especially insertion of negative or polarneutral residue) and at position 76 (substitution by negatively-chargedresidue) can often have stabilizing effect on the mutant protease andcan be used in combination. Mutations at for example one or more ofpositions 120, 170, 195, 235 and 251 have been found to be associatedwith increased enzyme activity. Even in cases where these lattermutations are associated individually with some loss of stability it canbe acceptable and useful to combine them with mutations at one or bothof positions 36 and 76.

Useful examples of such protease mutants include those having thefollowing mutations:

reference code amino acid substitution(s) S021) *36D S022) *36D +R170Y + G195E + K251E S023) *36D + H120D + R170Y + G195E + K235L S024)*36D + H120D + R170Y + G195E + K235L + K251E S025) *36D + H120D +G195E + K235L S235) *36D + N76D and S035) *36D + N76D + H120D + G195E +K235L.

Under some conditions, it can be advantageous to arrange a furthermutation by inserting a positive charge elsewhere, e.g. apositively-charged residue at position 213, e.g. T213K, in a mutantprotease having an insertion of a negatively-charged amino acid residueat position 36. This can increase the water solubility of the resultingmutant protease.

According to the invention, it is further preferred that the mutantsubtilisin enzyme represents a mutation of a parent enzyme selected fromsubtilisin BPN′, subtilisin amylosacchariticus, subtilisin 168,subtilisin mesentericopeptidase, subtilisin Carlsberg, subtilisin DY,subtilisin 309, subtilisin 147, thermitase, Bacillus PB92 protease andproteinase K, preferably subtilisin 309, subtilisin 147, subtilisinCarlsberg, aqualysin, Bacillus PB92 protease, Protease TW7 or ProteaseTW3.

Further preferred embodiments comprise subtilisin enzymes containing oneor more of the following mutations:

R10F, R10L, R10F+R45A+E89S+E136Q+R145A+D181N+R186P+E271Q,R10F+R19Q+E89S+E136Q+R145A+D181N+E271Q+R275Q, Q12K, Q12R,Q12K+P14D+T22K+N43R+Q59E+N76D+A98R+S99D+S156E+A158R+A172D+N173K+T213R+N248D+T255E+S256K+S259D+A272R,Q12R+P14D+T22R+N43R+Q59E+N76D+A98R+S99D+S156E+A158R+A172D+N173K+T213R+N248D+T255E+S256K+S259D+A272R,Q12K+P14D+T22K+T38K+N43R+Q59E+N76D+A98R+S99D+S156E+A158R+A172D+N173K+T213R+N248D+T255E+S256K+S259D+A272R,Q12R+P14D+T22R+T38R+N43R+Q59E+N76D+A98R+S99D+S156E+A158R+A172D+N173K+T213R+N248D+T255E+S256K+S259D+A272R,Q12K+P14D+T22K+T38K+N43R+Q59E+N76D+A98R+S99D+H120D+N140D+S141R+S156E+A158R+A172D+N173K+T213R+N248D+T255E+S256K+S259D+A272R,Q12R+P14D+T22R+T38R+N43R+Q59E+N76D+A98R+S99D+H120D+N140D+S141R+S156E+A158R+A172D+N173K+T213R+N248D+T255E+S256K+S259D+A272R,P14D, P14K, P14K+*36D, P14K+N218D, P14K+P129D, A15K, A15R, R19Q, T22K,T22R, K27R, K27V, D32*, *36D, *36D+R170Y+G195E+K251E,*36D+H120D+R170Y+G195E+K235L, *36D+H120D+R170Y+G195E+K235L+K251E,*36D+H120D+G195E+K235L, T38K, T38R, D41E, N43R, N43K, R45A, E53R, E53K,E53G+K235L, E54G, E54Y, Q59E,Q59E+N76D+A98R+S99D+S156E+A158R+A172D+N173K+T213R+N248D+T255E+S256K+S259D+A272R,D60N, N76D, E89S, E89S+K251N, Y91F, K94R, G97D, G97D+H120K, A98K, A98R,S99D, S99D+N140K, E112T, H120K, H120D, H120D+K235L, H120D+G195E+K235L,H120D+R170Y+G195E+K235L, H120D+R170Y+G195E+K235L+K251E, P129D, E136Q,E136K, E136R, E136Q+R10L, N140D, N140K, N140R, S141K, S141R, R145A,S156E, S156E+A158R+A172D+N173K, S156E+A158R+A172D+N173K+T213R,S156E+A158R+A172D+N173K+T213R+N248D+T255E+S256K+S259D+A272R, A158R,A158K, Y167V, R170Y, R170Y+G195E, R170Y+K251E, R170Y+G195E+K251E,R170Y+G195E+K235L, Y171E, Y171T, A172D, N173K, D181N, N184K, N184R,N185D, R186P, Y192V, Y192V,A, G195E, G195D, G195E+T213R, G195E+K251E,G195E+K235L, D197N, D197K, D197E, Q206D, Q206E, Y209L, T213R, T213K,Y214T, Y214S, N218D, N218S, K235L, K235R, K237R, W241Y,L, W241Y+H249R,W241L+H249R, N248D, H249R, K251R, K251E, K251N, T255E, S256R, S256K,S259L, S259D, Y263W, S265K, S265R, E271Q, E271G, E271G+K27V, E271Q,G,A272R, R275Q, D14K, D14K+D120K, D14K+D120K+D140K,D14K+D120K+D140K+D172K, K27D, K27D+D120K, E54T, E54Y, N97D, N97D+S98D,N97D+T213D, S98D, S98D+T213D, D120K, D140K, S156E, D172K, T213D andN218D.

Further specific preferred embodiments are mutated subtilisin proteasescomprising one or more of the following mutations:

S001) G195E

S002) G195D

S003) R170Y

S004) R170Y+G195E

S005) K251E

S006) H120D

S008) H120D+G195E

S009) T71D

S010) T71D+G195E

S011) R170Y+K251E

S012) R170Y+G195E+K251E

S013) T71D+R170Y+K251E

S014) T71D+R170Y+G195E+K251E

S015) K235L

S016) H120D+K235L

S017) H120D+G195E+K235L

S018) G195E+K251E

S019) H120D+R170Y+G195E+K235L

S020) H120D+R170Y+G195E+K235L+K251E

S021) *36D

S022) *36D+R170Y+G195E+K251E

S023) *36D+H120D+R170Y+G195E+K235L

S024) *36D+H120D+R170Y+G195E+K235L+K251E

S025) *36D+H120D+G195E+K235L

S026) E136R

S027) E89S

S028) D181N

S029) E89S+E136R

S030) E89S+D181N

S031) D197N+E271Q

S032) D197N

S033) E271Q

S035) *36D+N76D+H120D+G195E+K235L

S041) G195F

S201) N76D

S202) N76D+G195E

S203) N76D+R170Y+G195E

S204) H120D+G195E+K235L+K251E

S223) Q59E+N76D+A98R+S99D+T213K+K235L+N248D+T255E+S256K+S259D+A272R

S224)Q59E+N76D+A98R+S99D+H120D+N140D+S141R+K235L+N248D+T255E+S256K+S259D+A272R

S225)*36D+Q59E+N76D+A98R+S99D+R170Y+S156E+A158R+A172D+N173R+K235L+N248D+T255E+S256K+S259D+A272R

S226) *36Q

S227)*36D+Q59E+N76D+A98R+S99D+H120D+N140D+S141R+R170Y+G195E+K235L+N248D+T255E+S256K+S259D+A272R

S228)*36D+Q59E+N76D+A98R+S99D+H120D+N140D+S141R+S156E+A158R+A172D+N173K+K235L+N248D+T255E+S256K+S259D+A272R

S229)Q59E+N76D+A98R+S99D+H120D+N140D+S141R+S156E+A158R+A172D+N173K+K235L+N248D+T255E+S256K+S259D+A272R

S234) Q206D

S235) *36D+N76D

S242) *36Q+N76D+H120D+G195E+K235L

C001) D14K

C002) D120K

C003) D140K

C004) D14K+D120K

C005) K27D

C006) K27D+D120K

C008) D172K

C009) D14K+D120K+D140K

C010) D14K+D120K+D140K+D172K

C013) N97D

C014) S98D

C015) T213D

C017) S156E

C018) N97D+S98D

C019) N97D+T213D

C022) S98D+T213D

C028) N218D

C100) V51D

C101) E54T and

C102) E54Y.

The S series mutants preferably are based on subtilisin 309 as theparent subtilisin. The C series mutants preferably are based onsubtilisin Carlsberg.

In a further aspect of the invention, the above observations about thepI_(o) are further utilized in a method for determining or selecting theposition(s) and the amino acid(s) to be deleted, substituted or insertedfor the amino acid(s) in a parent enzyme, so that the net electrostaticcharge of the mutant enzyme has been changed in comparison to the NEC ofthe parent enzyme calculated at the same pH value.

Another way of expressing this principle covered by the invention isthat the position(s) and the amino acid(s) to be deleted, substituted orinserted for the amino acid(s) in said parent enzyme is selected in away whereby the total number of charges or total charge content (═TCC)and/or the NEC in a resulting mutant enzyme is changed in a way toprovide for a mutant protease having an isoelectric point shifted toachieve the pH for optimum wash performance of the enzyme, which pHoptimum should be as close as possible to the pH of the wash liquor,wherein said mutant protease is intended for use.

As indicated above, the pI_(o) of a macromolecule such as an enzyme iscalculated as the pH where the NEC of the molecule is zero. Theprocedure is exemplified in the examples below, but the principles aredescribed in more detail here.

pK values are assigned to each potentially charged amino acid residue.Then the ratio of the occurrence of an amino acid residue at a given pHin charged or uncharged form (charged/uncharged, C/U(i)) is calculatedfor both negative and positive charges by using formulas Ia and Ib:

C/U(i)=exp(ln ₁₀(pH−pK_(i)))(negative charge)  (Ia)

C/U(i)=exp(ln ₁₀(pK_(i)−pH))(positive charge)  (Ib).

According to the above formulas, if pH equals pK_(i), C/U(i) is equal to1.

The relative charge, Q_(r)(i), or charge contribution allocated to eachcharged residue is then calculated by using formulas IIa and IIb:

Q _(r)(i)=C/U(i)/(1+C/U(i))(negative charge)  (IIa)

Q _(r)(i)=−C/U(i)/(1+C/U(i))(positive charge)  (IIb).

The pH value where the sum of all the charge contributions from thecharged residues is equal to zero is found by iteration or throughinterpolation in a sufficiently dense pH-charge sum table.

Detergent Compositions Comprising the Mutant Enzymes

The present invention is also directed to the use of the mutant enzymesof the invention in cleaning and detergent compositions and tocompositions comprising the mutant subtilisin enzymes.

Such compositions comprise one or more of the mutant subtilisin enzymesaccording to the present invention alone or in combination with a lipaseor any of the usual components included in such compositions which arewell-known to the person skilled in the art.

Such components include builders, such as phosphate or zeolite builders,surfactants such as anionic, cationic or non-ionic, polymers such asacrylic or equivalent polymers, bleach systems such as perborate- oramino-containing bleach precursors or activators, structurants such assilicate structurants, alkali or acid to adjust pH, humectants and/orneutral inorganic salts.

In several useful embodiments the detergent compositions can beformulated as follows:

a) A detergent composition formulated as a detergent powder containingphosphate builder, anionic surfactant, nonionic surfactant, acrylic orequivalent polymer, perborate bleach precursor, amino-containing bleachactivator, silicate or other structurant, alkali to adjust to desired pHin use and neutral inorganic salt.

b) A detergent composition formulated as a detergent powder containingzeolite builder, anionic surfactant, nonionic surfactant, acrylic orequivalent polymer, perborate bleach precursor, amino-containing bleachactivator, silicate or other structurant, alkali to adjust to desired pHin use and neutral inorganic salt.

c) A detergent composition formulated as an aqueous detergent liquidcomprising anionic surfactant, nonionic surfactant, humectant, organicacid, caustic alkali, with a pH adjusted to a value between 9 and 10.

d) A detergent composition formulated as a nonaqueous detergent liquidcomprising a liquid nonionic surfactant consisting essentially of linearalkoxylated primary alcohol, triacetin, sodium triphosphate, causticalkali, perborate monohydrate bleach precursor and tertiary amine bleachactivator, with a pH adjusted to a value between about 9 and 10.

e) A detergent composition formulated as a detergent powder in the formof a granulate having a bulk density of at least 550 g/l, e.g. at least600 g/l, containing anionic and nonionic surfactants, e.g. anionicsurfactant and a mixture of nonionic surfactants with respectivealkoxylation degrees about 7 and about 3, low or substantially zeroneutral inorganic salt, phosphate builder, perborate bleach precursor,tertiary amine bleach activator, sodium silicate and minors andmoisture.

f) A detergent composition formulated as a detergent powder in the formof a granulate having a bulk density of at least 600 g/l, containinganionic surfactant and a mixture of nonionic surfactants with respectivealkoxylation degrees about 7 and about 3, low or substantially zeroneutral inorganic salt, zeolite builder, perborate bleach precursor,tertiary amine bleach activator, sodium silicate and minors andmoisture.

g) A detergent composition formulated as a detergent powder containinganionic surfactant, nonionic surfactant, acrylic polymer, fatty acidsoap, sodium carbonate, sodium sulphate, clay particles with or withoutamines, perborate bleach precursor, tertiary amine bleach activator,sodium silicate and minors and moisture.

h) A detergent composition formulated as a detergent (soap) barcontaining soap based on pan-saponified mixture of tallow and coconutoil, neutralized with orthophosphoric acid, mixed with protease, alsomixed with sodium formate, borax, propylene glycol and sodium sulphate,and then plodded on a soap production line.

i) An enzymatic detergent composition formulated to give a wash liquorpH of 9 or less when used at a rate corresponding to 0.4-0.8 g/lsurfactant.

j) An enzymatic detergent composition formulated to give a wash liquorpH of 8.5 or more when used at a rate corresponding to 0.4-0.8 g/lsurfactant.

k) An enzymatic detergent composition formulated to give a wash liquorionic strength of 0.03 or less, e.g. 0.02 or less, when used at a ratecorresponding to 0.4-0.8 g/l surfactant.

l) An enzymatic detergent composition formulated to give a wash liquorionic strength of 0.01 or more, e.g. 0.02 or more, when used at a ratecorresponding to 0.4-0.8 g/l surfactant.

m) A structured liquid detergent containing e.g. 2-15% nonionicsurfactant, 5-40% total surfactant comprising nonionic and optionallyanionic surfactant, 5-35% phosphate-containing ornon-phosphate-containing builder, 0.2-0.8% polymeric thickener, e.g.cross-linked acrylic polymer with m.w. over 10⁶, at least 10% sodiumsilicate, e.g. as neutral waterglass, alkali (e.g. potassium-containingalkali) to adjust to desired pH, preferably in the range 9-10 orupwards, e.g. above pH 11, with a ratio of sodium cation:silicate anion(as free silica) (by weight) of less than 0.7:1, and viscosity of 0.3-30Pa.s (at 20° C. and 20 reciproval secs).

For example, such detergents can contain about 5% nonionic surfactantC13-15 alcohol alkoxylated with about 5 EO groups per mole and about 2.7PO groups per mole, 15-23% neutral waterglass with 3.5 weight ratiobetween silica and sodium oxide, 13-19% KOH, 8-23% STPP, 0-11% sodiumcarbonate, 0.5% Carbopol 941™. Protease may be incorporated at forexample 0.5% of example S1.

The protease can be used in an amount ranging from e.g. about 0.0002 toabout 0.05 Anson units per gram of the detergent composition. Expressedin other units, the protease can also be included in the compositions inamounts of the order of from about 1 to about 100 GU/mg detergentformulation. Preferably, the amount ranges from 2 to 50 and particularlypreferably from 5 to 20 GU/mg.

A GU is a Glycine Unit and is defined as the proteolytic enzyme activitywhich produces an amount of NH₂-group equivalent to 1 micromole ofglycine during a 15-minute-incubation at 40° C. with N-acetyl casein assubstrate under standard conditions.

Detergent Compositions Comprising Mutant Enzymes and Lipases

It has surprisingly been found that a decrease in the isoelectric pointand hence net charge of a subtilisin type protease under washingconditions, can result in not only an improved wash performance of theenzyme but also an improved compatibility with lipase.

It also has been surprisingly found that compatibility of protease withlipase is influenced not only by the pI_(o) but also by the positions atwhich the charges are located relative to the active site of theprotease. The introduction of negative charge or removal of positivecharge closer to the active site gives stronger improvement ofcompatibility of protease with lipase.

Accordingly, certain embodiments of the invention provide enzymaticdetergent compositions comprising lipase and mutated subtilisinprotease, wherein the net molecular electrostatic charge of the mutatedprotease has been changed by insertion, deletion or substitution ofamino acid residues in comparison to the parent protease and wherein,there are, relative to said parent protease, fewer positively-chargedamino acid residue(s) and/or more negatively-charged amino acidresidue(s), whereby said subtilisin protease has an isoelectric pointlower than that of said parent protease.

One preferred class of lipases for such use originates in Gram-negativebacteria and includes e.g. lipase enzymes of the groups defined in EP 0205 208 and 0 206 390 (both to Unilever), (hereby incorporated byreference), including lipases immunologically related to those fromcertain Ps. fluorescens, P. gladioli and Chromobacter strains.

Preferred embodiments of mutantations of the subtilisin protease enzymefor use in conjunction with lipase include one or more mutations at thesite of an amino acid residue located within the range of about 15A-20Afrom the active site, especially for example at positions 170, 120 or195.

The lipase can usefully be added in the form of a granular composition(alternatively a solution or a slurry) of lipolytic enzyme with carriermaterial (e.g. as in EP 258068 (Novo Nordisk A/S) and Savinase® andLipolase®, products of Novo Nordisk A/S).

The added amount of lipase can be chosen within wide limits, for example50 to 30,000 LU/g per gram of the surfactant system or of the detergentcomposition, e.g. often at least 100 LU/g, very usefully at least 500LU/g, sometimes preferably above 1000, above 2000 LU/g or above 4000LU/g or more, thus very often within the range 50-4000 LU/g and possiblywithin the range 200-1000 LU/g. In this specification lipase units aredefined in EP 258068.

The lipolytic enzyme can be chosen from among a wide range of lipases,e.g. the lipases described in the following patent specifications, EP214761 (Novo Nordisk A/S), EP 0 258 068. Further preferred lipasesinclude lipases showing immunological cross-reactivity with antiseraraised against lipase from Thermomyces lanuginosus ATCC 22070, EP 0 205208 and EP 0 206 390 and lipases showing immunological cross-reactivitywith antisera raised against lipase from Chromobacter viscosum varlipolyticum NRRL B-3673, or against lipase from Alcaligenes PL-679, ATCC31371 and FERM-P 3783. Also preferred are the lipases described inspecifications WO 87/00859 (Gist-Brocades) and EP 0 204 284 (SapporoBreweries). Suitable in particular are for example the followingcommercially available lipase preparations: Novo Lipolase®, Amanolipases CE, P, B, AP, M-AP, AML and CES, and Meito lipases MY-30, OF andPL, also Esterase® MM, Lipozym®, SP225, SP285, Saiken lipase, Enzecolipase, Toyo Jozo lipase and Diosynth lipase (Trademarks).

Genetic engineering of the enzymes can be achieved by extraction of anappropriate lipase gene, e.g. the gene for lipase from Thermomyceslanuginosus or from a mutant thereof, and introduction and expression ofthe gene or derivative thereof in a suitable producer organism such asan Aspergillus. The techniques described in WO 88/02775 (Novo NordiskA/S), EP 0 243 338 (Labofina), EP 0 268 452 (Genencor) and notably EP 0305 216 (Novo Nordisk A/S) or EP 0 283 075 (Gist-Brocades) may beapplied and adapted.

Similar considerations apply mutatis mutandis in the case of otherenzymes, which may also be present. Without limitation: Amylase can forexample be used when present in an amount in the range about 1 to about100 MU (maltose units) per gram of detergent composition, (or 0.014-1.4,e.g. 0.07-0.7, KNU/g (Novo units)). Cellulase can for example be usedwhen present in an amount in the range about 0.3 to about 35 CEVU unitsper gram of the detergent composition.

The detergent compositions may furthermore include the following usualdetergent ingredients in the usual amounts. They may be built or unbuiltand may be of the zero-P type (i.e. not containing anyphosphorus-containing builders). Thus, the composition may contain inaggregate for example from 1-50%, e.g. at least about 5% and often up toabout 35-40% by weight, of one or more organic and/or inorganicbuilders. Typical examples of such builders include those alreadymentioned above and more broadly include alkali metal ortho, pyro andtripolyphosphates, alkali metal carbonates, either alone or in admixturewith calcite, alkali metal citrates, alkali metal nitrilotriacetates,carboxymethyloxysuccinates, zeolites, polyacetalcarboxylates and so on.

Furthermore, the detergent compositions may contain from 1-35% of ableaching agent or a bleach precursor or a system comprising bleachingagent and/or precursor with activator therefor. Further optionalingredients are lather boosters, foam depressors, anti-corrosion agents,soil-suspending agents, sequestering agents, anti-soil redepositionagents, perfumes, dyes, stabilizing agents for the enzymes and so on.

The compositions can be used for the washing of textile materials,especially but without limitation cotton and polyester-based textilesand mixtures thereof. Especially suitable are for example washingprocesses carried out at temperatures of about 60-65° C. or lower, e.g.about 30° C.-35° C. or lower. It can be very suitable to use thecompositions at a rate sufficient to provide about e.g. 0.4-0.8 g/lsurfactant in the wash liquor, although it is of course possible to uselesser or greater concentrations if desired. Without limitation it canbe stated that a use-rate e.g. from about 3 g/l to about 6 g/l of thedetergent formulation is suitable when the formulations provided in theExamples are used.

Method for Producing Mutations in Subtilisin Genes

Many methods for introducing mutations into genes are well known in theart. After a brief discussion of cloning subtilisin genes, methods forgenerating mutations in both random sites and specific sites within thesubtilisin gene will be discussed.

Cloning a Subtilisin Gene

The gene encoding subtilisin may be cloned from any Gram-positivebacteria or fungus by various methods well known in the art. First agenomic and/or cDNA library of DNA must be constructed using chromosomalDNA or messenger RNA from the organism that produces the subtilisin tobe studied. Then, if the amino acid sequence of the subtilisin is known,homologous, labelled oligonucleotide probes may be synthesized and usedto identify subtilisin-encoding clones from a genomic library ofbacterial DNA or from a fungal cDNA library. Alternatively, a labelledoligonucleotide probe containing sequences homologous to subtilisin fromanother strain of bacteria or fungus could be used as a probe toidentify subtilisin-encoding clones, using hybridization and washingconditions of lower stringency.

Yet another method for identifying subtilisin-producing clones involvesinserting fragments of genomic DNA into an expression vector, such as aplasmid, transforming protease-negative bacteria with the resultinggenomic DNA library and then plating the transformed bacteria onto agarcontaining a substrate for subtilisin, such as skim milk. Those bacteriacontaining subtilisin-bearing plasmid will produce colonies surroundedby a halo of clear agar due to the digestion of skim milk by excretedsubtilisin.

Generation of Random Mutations in the Subtilisin Gene

Once the subtilisin gene has been cloned into a suitable vector, such asa plasmid, several methods can be used to introduce random mutationsinto the gene.

One method would be to incorporate the cloned subtilisin gene as part ofa retrievable vector into a mutator strain of Eschericia coli.

Another method would involve generating a single stranded form of thesubtilisin gene and then annealing the fragment of DNA containing thesubtilisin gene with another DNA fragment so that a portion of thesubtilisin gene remained single stranded. This discrete, single strandedregion could then be exposed to any of a number of mutagenizing agents,including, but not limited to, sodium bisulfite, hydroxylamine, nitrousacid, formic acid or hydralazine. A specific example of this method forgenerating random mutations is described by Shortle and Nathans (1978,Proc. Natl. Acad. Sci. U.S.A., 75: 2170-2174). According to this method,the plasmid bearing the subtilisin gene would be nicked by a restrictionenzyme that cleaves within the gene. This nick would be widened into agap using the exonuclease action of DNA polymerase I. The resultingsingle-stranded gap could then be mutagenized using any one of the abovementioned mutagenizing agents.

Alternatively, the subtilisin gene from a Bacillus species including thenatural promoter and other control sequences could be cloned into aplasmid vector containing replicons for both E. coli and B. subtilis, aselectable phenotypic marker and the M13 origin of replication forproduction of single-stranded plasmid DNA upon superinfection withhelper phage IR1. Single-stranded plasmid DNA containing the clonedsubtilisin gene is isolated and annealed with a DNA fragment containingvector sequences but not the coding region of subtilisin, resulting in agapped duplex molecule. Mutations are introduced into the subtilisingene either with sodium bisulfite, nitrous acid or formic acid or byreplication in a mutator strain of E. coli as described above. Sincesodium bisulfite reacts exclusively with cytosine in a single-strandedDNA, the mutations created with this mutagen are restricted only to thecoding regions. Reaction time and bisulfite concentration are varied indifferent experiments so that from one to five mutations are created persubtilisin gene on average. Incubation of 10 μg of gapped duplex DNA in4 M Na-bisulfite, pH. 6.0, for 9 minutes at 37° C. in a reaction volumeof 400 μl, deaminates about 1% of cytosines in the single-strandedregion. The coding region of mature subtilisin contains about 200cytosines, depending on the DNA strand. Advantageously, the reactiontime is varied from about 4 minutes (to produce a mutation frequency ofabout one in 200) to about 20 minutes (about 5 in 200).

After mutagenesis, the gapped molecules are treated in vitro with DNApolymerase I (Klenow fragment) to make fully double-stranded moleculesand fix the mutations. Competent E. coli are then transformed with themutagenized DNA to produce an amplified library of mutant subtilisins.Amplified mutant libraries can also be made by growing the plasmid DNAin a Mut D strain of E. coli which increases the range for mutations dueto its error prone DNA polymerase.

The mutagens nitrous acid and formic acid may also be used to producemutant libraries. Because these chemicals are not as specific forsingle-stranded DNA as sodium bisulfite, the mutagenesis reactions areperformed according to the following procedure. The coding portion ofthe subtilisin gene is cloned in M13 phage by standard methods andsingle stranded phage DNA is prepared. The single-stranded DNA is thenreacted with 1 M nitrous acid pH. 4.3 for 15-60 minutes at 23° C. or 2.4M formic acid for 1-5 minutes at 23° C. These ranges of reaction timesproduce a mutation frequency of from 1 in 1000 to 5 in 1000. Aftermutagenesis, a universal primer is annealed to the M13 DNA and duplexDNA is synthesized using the mutagenized single-stranded DNA as atemplate so that the coding portion of the subtilisin gene becomes fullydouble-stranded. At this point, the coding region can be cut out of theM13 vector with restriction enzymes and ligated into an unmutagenizedexpression vector so that mutations occur only in the restrictionfragment (Myers et al., Science 229:242-257 (1985)).

Generation of Site Directed Mutations in the Subtilisin Gene

Once the subtilisin gene has been cloned and desirable sites formutation identified, these mutations can be introduced using syntheticoligonucleotides. These oligonucleotides contain nucleotide sequencesflanking the desired mutation sites; mutant nucleotides are insertedduring oligonucleotide synthesis. In a preferred method, a singlestranded gap of DNA bridging the subtilisin gene is created in a vectorbearing the subtilisin gene. The synthetic nucleotide bearing thedesired mutation is then annealed to a homologous portion of thesingle-stranded DNA. The remaining gap is then filled in by DNApolymerase I (Klenow fragment) and the construct is ligated using T4ligase. A specific example of this method is described in Morinaga etal. (1984, Biotechnology 2:646-639). According to Morinaga et al., afragment within the gene is removed using restriction endonuclease. Thevector/gene, now containing a gap, is then denatured and hybridized to avector/gene which, instead of containing a gap, has been cleaved withanother restriction endonuclease at a site outside the area involved inthe gap. A single-stranded region of the gene is then available forhybridization with mutated oligonucleotides, the remaining gap is filledin by the Klenow fragment of DNA polymerase I, the insertions areligated with T4 DNA ligase and after one cycle of replication, adouble-stranded plasmid bearing the desired mutation is produced. TheMorinaga method obviates the additional manipulation of constructing newrestriction sites and therefore facilitates the generation of mutationsat multiple sites. U.S. Pat. No. 4,760,025 by Estell et al., issued Jul.26, 1988, is able to introduce oligonucleotides bearing multiplemutations by performing minor alterations of the cassette. However, aneven greater variety of mutations can be introduced at any one time bythe Morinaga method, because a multitude of oligonucleotides of variouslengths can be introduced.

Expression of Subtilisin Mutants

According to the present invention, a mutated subtilisin gene producedby the methods described above or any other method known in the art, canbe expressed in enzyme form using an expression vector. An expressionvector generally falls under the definition of a cloning vector, sincean expression vector usually includes the components of a typicalcloning vector, namely, an element that permits autonomous replicationof the vector in a microorganism independent of the genome of themicroorganism, and one or more phenotypic markers for selectionpurposes. An expression vector includes control sequences encoding apromoter, operator, ribosome binding site, translation initiation signaland, optionally, a repressor gene or various activator genes. To permitthe secretion of the expressed protein, nucleotides encoding a “signalsequence” may be inserted prior to the coding sequence of the gene. Forexpression under the direction of control sequences, a target gene to betreated according to the invention is-operably linked to the controlsequences in the proper reading frame. Promoter sequences that can beincorporated into plasmid vectors and which can support thetranscription of the mutant subtilisin gene, include but are not limitedto the prokaryotic β-lactamase promoter (Villa-Kamaroff et al., 1978,Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731) and the tac promoter (DeBoeret al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25). Furtherreferences can also be found in “Useful proteins from recombinantbacteria” in Scientific American, 1980, 242:74-94.

According to one embodiment B. subtilis is transformed by an expressionvector carrying the mutated DNA. If expression is to take place in asecreting microorganism such as B. subtilis a signal sequence may followthe translation initiation signal and precede the DNA sequence ofinterest. The signal sequence acts to transport the expression productto the cell wall where it is cleaved from the product upon secretion.The term “control sequences” as defined above is intended to include asignal sequence, when it is present.

EXAMPLES Site Specific Mutation of the Subtilisin Gene Generates MutantsWith Useful Chemical Characteristics MATERIALS AND METHODS BacterialStrains

B. subtilis 309 and 147 are variants of Bacillus lentus deposited withthe NCIB and accorded accession numbers NCIB 10147 and NCIB 10309 anddescribed in U.S. Pat. No. 3,723,250, issued Mar. 27, 1973, which isincorporated herein by reference.

B. subtilis DN 497 is described in U.S. Ser. No. 039,298 filed Apr. 17,1987, corresponding to EP Publ. No. 242 220, which are also incorporatedherein by reference, and is an aro+ transformant of RUB 200 withchromosomal DNA from SL 438, a sporulation and protease deficient strainobtained from Dr. Kim Hardy of Biogen.

E. coli MC 1000 r^(−m)+ (Casadaban, M. J. and Cohen, S. N. (1980), J.Mol. Biol. 138: 179-207), was made r^(−m)+ by conventional methods andis also described in U.S. Ser. No. 039,298.

B. subtilis DB105 is described in Kawamura, F., Doi, R. H. (1984),Construction of a Bacillus subtilis double mutant deficient inextracellular alkaline and neutral proteases, J. Bacteriol. 160 (2),442-444.

Plasmids

pSX50 (described in U.S. Ser. No. 039,298 which is incorporated hereinby reference) is a derivative of plasmid pDN 1050 comprising thepromoter-operator P₁O₁, the B. pumilus xyn B gene and the B. subtilisxyl R gene.

pSX62 (described in U.S. Ser. No. 039,298, supra) is a derivative ofpSX52 (ibid), which comprises a fusion gene between the calf prochymosingene and the B. pumilus xyn B gene inserted into pSX50 (supra). pSX62was generated by inserting the E. coli rrn B terminator into pSX52behind the prochymosin gene.

pSX65 (described in U.S. Ser. No. 039,298, supra) is a derivative ofplasmid pDN 1050, comprising the promotor-operator P₂O₂ the B. pumilusxyn B gene and the B. subtilis xyl R gene.

pSX88 (described in unpublished International Patent Application No.PCT/DK 88/00002 (NOVO INDUSTRI A/S) which is incorporated herein byreference) is a derivative of pSX50 comprising the subtilisin 309 gene.

pSX92 was produced by cloning the subtilisin 309 into plasmid pSX62(supra) cut at Cla I and Hind III and Cla I filled prior to theinsertion of the fragments DraI-NheI and NheI-Hind III from the clonedsubtilisin 309 gene.

pSX93, shown in FIG. 3, is pUC13 (Vieira and Messing, 1982, Gene19::259-268) comprising a 0.7kb XbaI-Hind III fragment of the subtilisin309 gene including the terminator inserted in a polylinker sequence.

pSX119 (described in unpublished International Patent Application No.PCT/DK 88/00002, supra) is pUC13 harboring an EcoRI-XbaI fragment of thesubtilisin 309 gene inserted into the polylinker.

pSX120 is a plasmid where the HpaI-HindIII fragment with the subtilisin309 gene from pSX88 is inserted into EcoRV-HindIII on pDN 1681, in a waywhereby the protease gene is expressed by the amy M and amy Q promotors.pDN 1681 is obtained from pDN 1380 (Diderichsen, B. and Christiansen,L.: 1988, FEMS Microbiology Letters 56: 53-60) with an inserted 2.85 bpClaI fragment from B. amylolicuefaciens carrying the amy Q gene withpromotor (Takkinen et al.: 1983, J. Biol. Chem. 258: 1007ff).

pUC13 is described in Vieira, J. and Messing, J.: 1982, Gene 19:259-268.

pUC19 is described in Yanisch-Perron, C., Vieira, J. and Messing, J.,1985, Gene 33:103-119.

pUB110 is described in Lacey, R. W., Chopra, J. (1974), Genetic studiesof a multiresistant strain of Staphylococcus aureus, J. Med. Microbiol.7, 285-297 and in Zyprian, E. and Matzura, H. (1986), Characterizationof signals promoting gene expression on the Staphylococcus aureusplasmid pUB110 and development of a Gram-positive expression vectorsystem, DNA 5 (3), 219-225.

Genes

The genes for the various subtilisins were obtained as referenced in theliterature mentioned above. In particular the genes for the subtilisin309 and 147 enzymes were obtained as described in unpublishedInternational Patent Application No. PCT/DK 88/00002, supra.

Subtilisin Carlsberg Gene Construction

A synthetic gene was designed based on the coding sequence of the maturesubtilisin Carlsberg protease and its transcription terminator (Jacobs,M., Eliasson, M., Uhlen, M. and Flock, J.-I. (1985), Cloning, sequencingand expression of subtilisin Carlsberg from Bacillus licheniformis).Nucleic Acids Res. 13 (24), 8913-8926), linked to the pre and pro codingsequences of the subtilisin BPN′ protease (Wells, J. A., Ferrari, E.,Henner, D. J., Estell, D. A. and Chen, E. Y. (1983), Cloning, sequencingand secretion of Bacillus amyloliquefaciens subtilisin in Bacillussubtilis, Nucleic Acids Res. 11 (22), 7911-7925). The gene wassubdivided into seven fragments in length ranging from 127 to 313 basepairs, each fragment built up from chemically synthesized oligos of 16to 77 nucleotides. The overlap between the oligos of the two strands wasoptimized in order to facilitate a one step annealing of each fragment(Mullenbach, G. T., Tabrizi, A., Blacher, R. W. and Steimer, K. S.(1986), Chemical synthesis and expression in Yeast of a gene encodingconnective tissue activating peptide-III, J. Biol. Chem. 261 (2),719-722). Each fragment was assembled and cloned in an E. Coli cloningand sequencing vector. Sequence analysis of these cloned fragments wasperformed to confirm the correctness of the sequence of each fragment.Then all of the fragments were assembled and cloned in the vector pUB110(Lacey, R. W. and Chopra, J. (1974), Genetic studies of a multiresistantstrain of Staphylococcus aureus, J. Med. Microbiol. 7, 285-297) andbrought into B. subtilis DB105 (Kawamura, F. and Doi, R. H. (1984),Construction of a Bacillus subtilis double mutant deficient inextracellular alkaline and neutral proteases, J. Bacteriol. 160 (2),442-444). Transcription of the gene was initiated by the HpaII promotorof the pUB110 plasmid vector (Zyprian, E. and Matzura, H. (1986),Characterization of signals promoting gene expression on theStaphylococcus aureus plasmid pUB110 and development of a Gram-positiveexpression vector system, DNA 5 (3), 219-225). In the process of thegene construction it turned out that the longest fragment (#5; 313 basepairs long) needed further fragmentation (fragments #8 and #9) in orderto avoid problems with the assembly of this rather long fragment.

The amino acid sequence deduced from the nucleotide sequence differsfrom the earlier published subtilisin Carlsberg sequence at positions129, 157, 161 and 212 (Smith, E. L., DeLange, R. J., Evans, W. H.,Landon, W. and Markland, F. S. (1968), Subtilisin Carlsberg V. Thecomplete sequence: comparison with subtilisin BPN′; evolutionaryrelationships., J. Biol. Chem. 243 (9), 2184-2191). A fifth alterationreported by Jacobs et al. (1985) could not be confirmed in the clone ofthe Carlsberg gene described here.

Computation of Isoelectric Point (pI_(o))

The calculation of the isoelectric point of subtilisin 309 wild typeenzyme (S000) is exemplified below in order to demonstrate the procedureused. The same procedure is applicable to the computation of any enzyme,whether it is a mutant enzyme or not.

pK values were assigned to each potentially charged amino acid residue(Tyr, Asp, Glu, Cys, Arg, His, Lys, N-terminal, C-terminal, Ca²⁺). Inthis case the environment was taken into consideration, wherebydifferent pK values are used for the same amino acid residue dependenton its neighbors. The assigned values are indicated in Table II.

The ratio of the occurrence of an amino acid residue at a given pH incharged or uncharged form (charged/uncharged, C/U(i)) was thencalculated for both negative and positive charge, by using formulas Iaand Ib, respectively. In Table II, this ratio is only indicated for pHequal to pI_(o).

Subsequently, the relative charge, Q_(r)(i) or charge contributionallocated to each charged residue was calculated by using formulas IIaand IIb.

The pH value where the sum of all the charge contributions from thecharged residues is equal to zero was found by iteration.

TABLE II Calculation of isoelectric point for: S000 Subtilisin 309 Q_(r)(i) Number of pH = Q_(r) (i) Q_(r) (i) Residue pK Residues C/U (i)* 8.3pH = 10.0 pH = pI_(o) Tyr 9.9 3 2.51E−02 −0.07 −1.67 −1.77 Tyr 11.6 25.01E−04 0.00 −0.05 −0.06 Tyr 12.5 2 6.31E−05 0.00 −0.01 −0.01 Asp 3.5 56.31E+04 −5.00 −5.00 −5.00 Glu 4 5 2.00E+04 −5.00 −5.00 −5.00 C-term 3 12.00E+05 −1.00 −1.00 −1.00 (Arg) Cys 9.3 0 1.00E−01 0.00 0.00 0.00 Arg12.8 8 3.16E+04 8.00 7.99 7.99 His 6.4 7 1.26E−02 0.09 0.00 0.00 Lys 105 5.01E+01 4.90 2.50 2.34 Calcium 20 1.25 5.01E+11 2.50 2.50 2.50 N-term8 1 5.01E−01 0.33 0.01 0.01 (Ala) Net 4.75 0.27 0.00 charge Thecalculated isoelectric point is 10.06. * E−02 = 10⁻²

As indicated above and in Table II, the pK value assigned to each aminoacid was different taking local variations in the environment intoconsideration. This results only in an enhanced precision in thecalculation, but experience has shown that constant estimated pK valuesare helpful in showing in what direction the pI_(o), for a given mutantenzyme will move in comparison to the pI_(o) of the parent enzyme. Thisis indicated in Table III, where pI_(o) values for estimated pK valuesare indicated.

In order to compare various enzymes and mutant enzymes, washing testsdescribed in detail below have been performed. In Table III below,results from these tests using parent enzyme and mutant enzymes fromsubtilisin 309 (designated S000, etc.) and subtilisin carlsberg(designated C000, etc.) have been tabulated in order to demonstrate thecorrelation between pI_(o) and wash performance at different pH valuesof the wash liquor used. In the washing tests, a low salt liquiddetergent formulation of pH 8.3 according to detergent example D7 and anormal salt powder detergent of pH 10.2 according to detergent exampleD2 were used.

In Table III, the results are compared to the wild type enzymes (S000and C000, respectively). In addition, the calculated and observedpI_(o)'s for the enzymes are indicated.

TABLE III Comparative washing tests at different pH values ImprovementFactor pI_(o) Detergent pH Mutant calculated observed 8.3 10.2 S00010.02  9.7 1   1   S001 9.86 9.4 2.2 1   S003 9.86 9.4 2.0 1   S004 9.689.1 3.9 1   S005 9.71 9.1 1.5 1   S012 9.09 8.8 5.0 0.6 S019 9.09 8.55.8 0.6 S020 6.71 7.9 8.8 0.5 S021 9.85 — 1.8 0.7 S022 8.07 — 9.0 0.3S023 8.05 — 9.8 0.2 S024 6.86 — 9.0 0.2 S025 8.94 — 6.9 0.6 S027 10.28 — 0.4 1.0 S028 10.28  — 0.9 1.0 S031 10.53  — 0.4 0.7 S032 10.28  — 0.7— S033 10.28  — 0.4 — S035 8.07 — 8.0 0.6 S201 9.85 — 2.0 0.7 S202 9.62— 4.3 0.9 S203 9.27 — 9.0 0.5 C000 8.87 — 1   1   C001 9.38 — 0.2 1.5C002 9.38 — 0.8 1.9 C003 9.38 — 0.4 1.1 C004 9.64 — 0.2 1.8 C008 9.38 —0.2 1.5

From Table III, it is seen that shifting the pI_(o) to lower values(S-series) provides for an improvement in wash performance at low pH(pH=8.3), whereas an upward shift in pI_(o) (C-series) provides for animprovement in wash performance at high pH (pH=10.2).

The concept of isoelectric point has thus been found to be very usefulin selecting the positions of the amino acids in the parent enzyme whichshould be changed.

It has generally been found that mutations should be performed in codonscorresponding to amino acids situated at or near to the surface of theenzyme molecule thereby retaining the internal structure of the parentenzyme as much as possible.

Purification of Subtilisins

The procedure relates to a typical purification of a 10 liter scalefermentation of the Subtilisin 147 enzyme, the Subtilisin 309 enzyme ormutants thereof.

Approximately 8 liters of fermentation broth were centrifuged at 5000rpm for 35 minutes in 1 liter beakers. The supernatants were adjusted topH 6.5 using 10% acetic acid and filtered on Seitz Supra S100 filterplates.

The filtrates were concentrated to approximately 400 ml using an AmiconCH2 Å UF unit equipped with an Amicon S1Y10 UF cartridge. The UFconcentrate was centrifuged and filtered prior to absorption at roomtemperature on a Bacitracin affinity column at pH 7. The protease waseluted from the Bacitracin column at room temperature using 25%2-propanol and 1 M sodium chloride in a buffer solution with 0.01dimethylglutaric acid, 0.1 M boric acid and 0.002 M calcium chlorideadjusted to pH 7.

The fractions with protease activity from the Bacitracin purificationstep were combined and applied to a 750 ml Sephadex G25 column (5 cmdia.) equilibrated with a buffer containing 0.01 dimethylglutaric acid,0.2 M boric acid and 0.002 M calcium chloride adjusted to pH 6.5.

Fractions with proteolytic activity from the Sephadex G25 column werecombined and applied to a 150 ml CM Sepharose CL 6B cation exchangecolumn (5 cm dia.) equilibrated with a buffer containing 0.01 Mdimethylglutaric acid, 0.2 M boric acid and 0.002 M calcium chlorideadjusted to pH 6.5.

The protease was eluted using a linear gradient of 0-0.1 M sodiumchloride in 2 liters of the same buffer (0-0.2 M sodium chloride in caseof sub 147).

In a final purification step protease containing fractions from the CMSepharose column were combined and concentrated in an Amiconultrafiltration cell equipped with a GR81PP membrane (from the DanishSugar Factories Inc.).

Subtilisin 309 and mutants

Gly 195 Glu (G195E (S001));

Arg 170 Tyr (R170Y (S003));

Arg 170 Tyr+Gly 195 Glu (R170Y+G195E (S004));

Lys 251 Glu (K251E (S005));

His 120 Asp (H120D (S006));

Arg 170 Tyr+Gly 195 Glu+Lys 251 Glu (R170Y+G195E+K251E (S012));

Lys 235 Leu (K235L (S015)); His 120 Asp+Gly 195 Glu+Lys 235 Leu

(H120D+G195E+K235L (S017)); His 120 Asp+Arg 170 Tyr+Gly 195 Glu+Lys 235Leu (H120D+R170Y+G195E+K235L (S019)); and

His 120 Asp+Arg 170 Tyr+Gly 195 Glu+Lys 235 Leu+Lys 251 Glu(H120D+R170Y+G195E+K235L+K251E (S020));

were purified by this procedure.

Purification of (Mutant) Subtilisin Carlsberg Proteases

Fermentation media were either directly applied on a bacitracin affinitycolumn (5 cm diam * 15 cm; equilibrated with 10 mM Tris/HCl buffer pH7.9; flow rate approx. 500 ml/h) or concentrated to 500 ml by means of aNephross Andante H. F. dialyzer (Organon Technika) using a back pressureof 10-12 p.s.i. and demineralized water in the outer circuit. In thelatter case, the protease was precipitated from the concentrate byadding 600 g/l ammonium sulphate. The precipitate was collected by meansof centrifugation and redissolved in approx. 500 ml demineralized water.The ammonium sulphate was removed from the protease solution using thesame dialyzer as described above. The final volume was approx. 300 ml,while the pH was adjusted to pH 6.0. The protease was eluted from thebacitracin columns (mentioned above) using a 10 mM Tris buffer (pH 7.9)containing 2.7 M NaCl and 18% isopropanol.

After dialysis of bacitracin-purified or concentrated protease material,further purification was accomplished by application on a CM-Trisacrylion exchange column (5 cm. diam * 15 cm; equilibrated with 0.03M sodiumphosphate pH 6.0) using a flow rate of 200 ml/h. The protease was elutedfrom the column with a linear gradient from 0 to 0.3 M NaCl (2*500 ml)in the phosphate buffer. Fractions containing protease activity werepooled and stored at −20° C. in the presence of buffer salts afterfreeze-drying.

Oligonucleotide Synthesis

All mismatch primers were synthesized on an Applied Biosystems 380 A DNAsynthesizer and purified by polyacrylamide gel electrophoresis (PAGE).

Assay for Proteolytic Activity

The proteolytic activity of the mutant enzymes was assayed in order todetermine how far the catalytic activity of the enzyme was retained. Thedeterminations were performed by the dimethyl casein (DMC) methoddescribed in NOVO Publication AF 220-gb (or later editions), availablefrom Novo-Nordisk A/S, Bagsvard, Denmark, which is incorporated hereinby reference.

Assays for Wash Performance

A:

Test cloths (2.2 cm×2.2 cm, approximately 0.1 g) were produced bypassing desized cotton (100% cotton, DS 71) cloth through the vessel ina Mathis Washing and Drying Unit type TH (Werner Mathis AG, Zurich,Switzerland) containing grass juice.

Finally, the cloth was dried in a strong air stream at room temperature,stored at room temperature for 3 weeks, and subsequently kept at −18° C.prior to use.

All tests were performed in a model miniwash system. In this system, sixtest cloths were washed in a 150 ml beaker containing 60 ml of detergentsolution. The beakers were kept in a thermostat water bath at 30° C.with magnetic stirring.

The following standard liquid detergent was used:

AE, Berol 160 15% LAS, Nasa 1169/F 10% Coconut fatty acid 9% Oleic acid1% Triethanolamine 9% Glycerol 1.5% Ethanol 8% Tri.Na.Citrat.2H₂O 0.1%CaCl.2H₂O 0.1% NaOH 1% Water from LAS 23.3% Water from glycerol 1.5%Water added 34.9%

The percentages given are the percentage of active content.

The pH was adjusted with 1 N NaOH to 8.14. The water used was ca. 6° dH(German Hardness).

Tests were performed at enzyme concentrations of: 0, 1.0 mg enzymeprotein/1 and 10.0 mg enzyme protein/1, and two independent sets oftests were performed for each of the mutants. The results shown in thefollowing are means of these tests.

The washings were performed for 60 minutes and the cloths were thenflushed in running tap-water for 25 minutes in a bucket.

The cloths were then air-dried overnight (protected against daylight)and the remission, R, was determined on an ELREPHO 2000 photometer fromDatacolor S. A., Dietkikon, Switzerland at 460 nm.

As a measure of the wash performance, differential remission, delta R,was used. Differential remission is equal to the remission after washwith enzyme added minus the remission after wash with no enzyme added.

B:

The wash performance of various mutants was tested against grass juicestained cotton cloths according to the method described above.

2.0 g/l of a commercial US liquid detergent was used.

The detergent was dissolved in a 0.005 M ethanolamine buffer inion-exchanged water. The pH was adjusted to pH 8.0, 9.0, 10.0 and 11.0respectively with NaOH/HCl.

The temperature was kept at 30° C. isothermic for 10 min.

The mutants were dosed at 0.25 mg enzyme protein/1 each.

C:

Washing tests using the detergent compositions exemplified in thedetergent examples below were performed in a mini washer utilizingcotton based test cloths containing pigments, fat and protein (casein).The conditions were:

a) 2 g/l detergent D3 in 6°fH (French hardness) water at pH 8.3 or

b) 5 g/l detergent D2 in 15°fH water at pH 10.2.

After rinsing and drying, reflection at 460 nm was measured.

The improvement factor was calculated from a dose-response curve andrelates to the amount of enzyme needed for obtaining a given delta Rvalue in comparison to the wild type enzyme in question (S000 and C000),meaning that an improvement factor of 2 indicates that only half theamount of enzyme is needed to obtain the same delta R value.

The results of these tests are shown in Table III above.

D:

Experimental tests of lipase stability were carried out for exampleusing the following materials:

1 LU/ml Pseudomonas cepacia lipase was incubated in wash liquor of eachof two types, O and W (described below). Aliquots were taken atintervals and tested for lipase activity. Parallel incubations werecarried out without protease or with protease of various types as notedbelow, to test the effect of the protease on the retention of lipaseactivity. Wild-type proteases were tested at 20 GU/ml, mutated proteaseswere tested at 0.5 microgram/ml.

Detergent Compositions Comprising Enzyme Variants

The invention is illustrated by way of the following non-limitingExamples:

Detergent D1:

A detergent powder is formulated to contain: total active detergentabout 16%, anionic detergent about 9%, nonionic detergent about 6%,phosphate-containing builder about 20%, acrylic or equivalent polymerabout 3.5% (alternatively down to about 2%), perborate bleach precursorabout 6-18%, alternatively about 15-20%, amino-containing bleachactivator about 2%, silicate or other structurant about 3.5%,alternatively up to about 8%, enzyme of about 8 glycine units/mgactivity, with alkali to adjust to desired pH in use, and neutralinorganic salt and enzymes (about 0.5% each enzyme).

The anionic detergent is a mixture of sodium dodecyl-benzene sulphonate,alternatively sodium linear alkyl-benzene-sulphonate, 6% and primaryalkyl sulphate 3%. The nonionic detergent is an ethoxylate of an approx.C13-C15 primary alcohol with 7 ethoxylate residues per mole. Thephosphate builder is sodium tripolyphosphate. The polymer is polyacrylicacid, alternatively acrylic/maleic copolymer. The perborate bleachprecursor is sodium tetraborate tetrahydrate or monohydrate. Theactivator is tetraacetylethylenediamine. The structurant is sodiumsilicate. The neutral inorganic salt is sodium sulphate.

The enzymes comprise protease according to Mutant S001, alternativelyprotease S003, S004, S005, C001, C002, C003, C004, C005, C008, S015,S017, S021, S226, S223, S224 or S225.

Detergent D1a:

A detergent powder is formulated to contain: total active detergentabout 15%, anionic detergent about 7%, nonionic detergent about 6%,phosphate-containing builder about 25%, acrylic or equivalent polymerabout 0.5%, perborate bleach precursor about 10%, amino-containingbleach activator about 2%, silicate or other structurant about 6%,protease enzyme of about 8 glycine units/mg grade, with alkali to adjustto desired pH in use, and neutral inorganic salt and enzymes (about 0.5%each enzyme).

The anionic detergent is sodium linear alkyl-benzene-sulphonate. Thenonionic detergent is an ethoxylate of an approx. C13˜C15 primaryalcohol with 7 ethoxylate residues per mole or a mixture of this withthe corresponding alcohol ethoxylated to the extent of 3 residues permole. The phosphate builder is sodium tripolyphosphate. The perborate orperacid bleach precursor is sodium tetraborate tetrahydrate. Theactivator is tetraacetylethylenediamine. The structurant is sodiumsilicate. The neutral inorganic salt is sodium sulphate. The enzymescomprise protease according to Mutant S001, alternatively S003, S004,S005, C001, C002, C003, C004, C005, C008, S015, S017, S021 or S226.

Detergent D2:

A detergent powder is formulated to contain: total active detergentabout 16%, anionic detergent about 9%, nonionic detergent about 6%,zeolite-containing builder about 20%, acrylic or equivalent polymerabout 3.5%, perborate bleach precursor about 6-18%, amino-containingbleach activator about 2%, silicate or other structurant about 3.5%,alternatively down to about 2.5%, enzyme of about 8 (alternatively about15) glycine units/mg grade, with alkali to adjust to desired pH in use,and neutral inorganic salt and enzymes (about 0.5% each enzyme).

The anionic detergent is a mixture of sodium dodecyl-benzene sulphonate,alternatively sodium linear alkyl-benzene-sulphonate, 6% and primaryalkyl sulphate 3%. The nonionic detergent is an ethoxylate of an approx.C13-C15 primary alcohol with 7 ethoxylate residues per mole. The zeolitebuilder is type A zeolite. The polymer is polyacrylic acid. Theperborate bleach precursor is sodium tetraborate tetrahydrate ormonohydrate. The activator is tetraacetylethylenediamine. Thestructurant is sodium silicate. The neutral inorganic salt is sodiumsulphate. The enzymes comprise protease according to Mutant S001,alternatively S003, S004, S005, C001, C002, C003, C004, C005, C008,S015, S017, S021 or S226.

Detergent D2a:

A detergent powder is formulated to contain: total active detergentabout 14%, anionic detergent about 7%, nonionic detergent about 7%,zeolite-containing builder about 25%, acrylic or equivalent polymerabout 3%, perborate or peracid bleach precursor about 10%,amino-containing bleach activator about 2%, silicate or otherstructurant about 0.5%, enzyme of about 6 glycine units/mg grade, withalkali to adjust to desired pH in use, and neutral inorganic salt andenzymes (about 0.5% each enzyme).

The anionic detergent is sodium linear alkyl-benzene-sulphonate, thenonionic detergent is a mixture of ethoxylates of an approx. C13-C15primary alcohol with 7 and 3 ethoxylate residues respectively per mole.The zeolite builder is type A zeolite. The polymer is an acrylic/maleiccopolymer. The perborate bleach precursor is sodium tetraboratemonohydrate. The activator is tetraacetylethylenediamine. Thestructurant is sodium silicate. The neutral inorganic salt is sodiumsulphate. The enzymes comprise protease according to Mutant S001,alternatively S003, S004, S005, C001, C002, C003, C004, C005, C008,S015, S017, S021 or S226.

Detergent D3:

An aqueous detergent liquid is formulated to contain:Dodecylbenzene-sulphonic acid 16%, C12-C15 linear alcohol condensed with7 mol/mol ethylene oxide 7%, monoethanolamine 2%, citric acid 6.5%,sodium xylenesulphonate 6%, sodium hydroxide about 4.1%, protease 0.5%,minors and water to 100%. The pH is adjusted to a value between 9 and10. The enzyme is a protease according to Mutant S020, alternativelyS019, S012, S004, S001, S003, S005, S015, S017, S021, S022, S025, S035,S201, S223-6 or S235.

Detergent D4:

A nonaqueous detergent liquid is formulated using 38.5% C13-C15 linearprimary alcohol alkoxylated with 4.9 mol/mol ethylene oxide and 2.7mol/mol propylene oxide, 5% triacetin, 30% sodium triphosphate, 4% sodaash, 15.5% sodium perborate monohydrate containing a minor proportion ofoxoborate, 4% TAED, 0.25% EDTA of which 0.1% as phosphonic acid, Aerosil0.6%, SCMC 1% and 0.6% protease. The pH is adjusted to a value between 9and 10, e.g. about 9.8. The enzyme comprises protease according toMutant S001, alternatively S003, S004, S021, S035, S201, S225, S226 orS235.

Detergent D5:

A detergent powder is formulated in the form of a granulate having abulk density of at least 600 g/l, containing about 20% by weightsurfactant of which about 10% is sodium dodecylbenzene sulphonate andthe remainder is a mixture of Synperonic A7 and Synperonic A3 (about5.5% to 4.5%) and zero neutral inorganic salt (e.g. sodium sulphate),plus phosphate builder about 33%, sodium perborate tetrahydrate about16%, TAED activator about 4.5%, sodium silicate about 6% and minorsincluding sodium carbonate about 2% and moisture content about 10%.Enzymes (about 0.5% each enzyme) are included. The enzyme comprisesprotease according to Mutant S001, alternatively S003, S004, S005, C001,C002, C003, C004, C005, C008, S223, S224, S225, S226 or S235.

Detergent D6:

A detergent powder is formulated in the form of a granulate having abulk density of at least 600 g/l, alternatively about 550 g/l,containing about 20%, alternatively down to about 16%, by weightsurfactant of which about 9%, alternatively about 7%, is sodiumdodecylbenzene sulphonate, alternatively sodium linear alkyl benzenesulphonate and the remainder is a mixture of Synperonic A7 andSynperonic A3 (or similar ethoxylates) (respectively about 5% & 6%,alternatively about 4% and 7%) and zero neutral inorganic salt (e.g.sodium sulphate), plus zeolite builder about 30%, alternatively about25%, sodium perborate tetrahydrate, alternatively monohydrate, about 14%or 15%, TAED activator about 3.6% and minors including sodium carbonateabout 9%, or up to 15%, Dequest® 2047 about 0.7% and moisture contentabout 10%. Enzymes (about 0.5% each enzyme, or about 0.2% lipase andabout 0.7% protease) are included. The enzyme comprises proteaseaccording to Mutant S001, alternatively S003, S004, S005, C001, C002,C003, C004, C005, C008, S223, S224, S225, S226 or S235.

Detergent D6a:

A detergent powder is formulated in the form of a granulate having abulk density of at least 600 g/l, containing about 15% by weightsurfactant of which about 7% is sodium linear alkyl benzene sulphonate,2% primary alcohol sulphate and the remainder Synperonic A7 or similarethoxylate and zero neutral inorganic salt (e.g. sodium sulphate), pluszeolite builder about 22%, sodium perborate tetrahydrate about 15%, TAEDactivator about 7% and minors including sodium carbonate about 15t,Dequest® 2047 about 0.7% and moisture content about 10%. Enzymes (about1.2%) include protease according to Mutant S001, alternatively S003,S004, S005, C001, C002, C003, C004, C005, C008, S223, S224, S225, S226or S235.

Detergent D7:

A detergent powder is formulated to contain: Dodecylbenzenesulphonicacid 6%, C12-C15 linear alcohol condensed with 7 mol/mol ethylene oxide5%, fatty acid soap 3%, Sokolan® CP5 polymer 3%, zeolite A 22%, sodiumcarbonate 10%, sodium sulphate 17%, clay particles 8%, sodium perboratetetrahydrate 13%, tetraacetylethylenediamine 2%, protease 0.5%, minorsand water to 100%. The pH is adjusted to a value between 9 and 10. Theprotease enzyme comprises protease according to Mutant S001,alternatively S003, S004, S005, C001, C002, C003, C004, C005, C008,S223, S224, S225, S226 or S235.

Detergent D8:

A detergent (soap) bar according to an embodiment of the invention isformulated as follows: soap based on pan-saponified 82% tallow, 18%coconut oil, neutralized with 0.15% orthophosphoric acid, mixed withprotease (about 8 GU/mg of the bar composition) and with sodium formate2%, borax 2%, propylene glycol 2% and sodium sulphate 1%, is thenplodded on a soap production line. The protease enzyme comprisesprotease according to Mutant S001, alternatively S003, S004, S005, C001,C002, C003, C004, C005, C008, S021, S025, S035, S201, S202, S223, S224,S225, S226 or S235.

Detergent D9:

Structured liquid detergents can for example contain, in addition to aprotease as described herein, 2-15% nonionic surfactant, 5-40% totalsurfactant, comprising nonionic and optionally anionic surfactant, 5-35%phosphate-containing or non-phosphate containing builder, 0.2-0.8%polymeric thickener, e.g. cross-linked acrylic polymer with m.w. over10⁶, at least 10% sodium silicate, e.g. as neutral waterglass, alkali(e.g. potassium-containing alkali) to adjust to a desired pH, preferablyin the range 9-10 or upwards, e.g. above pH 11, with a ratio sodiumcation: silicate anion (as free silica) (by weight) of less than 0.7:1and viscosity of 0.3-30 Pas (at 20° C. and 20^(s−1)).

Suitable examples contain about 5% nonionic surfactant C13-15 alcoholalkoxylated with about 5 EO groups and about 2.7 PO groups per mole,15-23% neutral waterglass with 3.5 weight ratio between silica andsodium oxide, 13-19% KOH, 8-23% STPP, 0-11% sodium carbonate, 0.5%Carbopol® 941.

Protease (e.g. 0.5%) includes Mutant S001, alternatively S021, S025,S035, S201, S202, S223, S224, S225, S226 or S235.

Detergent D10:

A structured, viscous, aqueous liquid detergent suitable for laundry useis formulated as follows (% by weight):

Citric acid 2.5 Borax (10 aq) 4   NaOH 2   Glycerol 5   C14-C15 Linearalkyl-benzene- 6.5 sulphonate, or C14-15 primary alcohol sulphateSynperonic A3 1.2 Nonionic C12-C15 3EO Synperonic A7 3.6 NonionicC12-C15 7EO Zeolite 20   Protease 0.5 Amylase (Termamyl ® 300LDX) 0.2minors and water to 100%

The pH can be adjusted to a value between 9 and 10. The enzyme comprisesprotease Mutant S020, alternatively S019, S012, S004, S001, S003, S005,S021, S035, S201, S223-6 or S235.

Detergent D11:

An isotropic aqueous liquid detergent suitable for laundry use isformulated as follows (% by weight):

Citric acid 2   Boric acid 1   NaOH 3   KOH 4.5 Glycerol 10   Ethanol6.5 Nonionic surfactant 10   (C12-alcohol 6.5EO ethoxylate groups/mol)or sodium primary alcohol sulphate Oleic acid 16   Coconut oil (C12)soap 1.1 Protease 0.5 minors and water to 100%

The pH can be adjusted to a value between 9 and 10. The enzyme comprisesprotease Mutant S020, alternatively S019, S012, S004, S001, S003, S005,S021, S025, S035, S201, S223-6 or S235.

Detergent D12:

An aqueous liquid detergent composition is formulated to contain:

sodium alkyl-benzene-sulphonate 14.5 C18 sodium soap 2   Nonionicdetergent (C12-15 6EO) 9   Fatty acid (oleic acid) 4.5 sodium alkenylsuccinate 11   propanediol 1.5 ethanol 3.6 sodium citrate 3.2 Complexingagent e.g. Dequest 2060 0.7 Protease 0.5 Amylase 0.1 Sodium chloride 0.5minors and water to 100%

The pH can be adjusted to a value between 9 and 10. The enzyme comprisesprotease Mutant S020, alternatively S019, S012, S004, S001, S003, S005,S021, S025, S035, S201, S202, S223-6 or S235.

Detergent D13:

An aqueous liquid detergent composition is formulated to contain:

sodium alkyl-benzene-sulphonate 14.5 sodium alkyl-benzene-sulphonate 8  nonionic detergent 6.5EO 10   Oleic diethylamide 10   Fatty acid(C12/C18 75:25) 18   sodium citrate 1   triethanolamine 5   propanol 7  ethanol 5   Dequest 2060 0.5 Protease 0.5 Amylase 0.1 minors and waterto 100%

The pH can be adjusted to a value between 9 and 10. The enzyme comprisesprotease Mutant S020, alternatively S019, S012, S004, S001, S003, S005,S021, S025, S035, S201, S202, S223-6 or S235.

Detergent D14:

A non-aqueous liquid detergent composition is formulated to contain (%by weight):

Liquid nonionic detergent (C10-12, 6.2EO) 41% triacetin 5   linearalkylbenzenesulphonic acid 6   magnesium oxide stabilizer 1   Sodiumcarbonate builder/base 18   Calcium carbonate builder 8   bleachactivator TAED 3.5 bleach precursor perborate monohydrate 10.5 partly-hydrophobic silica 2   protease 0.4 lipase (Lipolase ®) 3  minors or additional to 100% liquid nonionic surfactant (no water)

In formulating this composition, the liquid nonionic surfactant andtriacetin are added first, followed by the magnesium oxide, then theother ingredients except enzyme. The mixture is milled in a colloid milland cooled and finally the enzyme(s) and any other heat-sensitive minorsare added.

The enzyme comprises protease Mutant S020, alternatively S019, S012,S004, S001, S003, S005, S021, S025, S035, S201, S202, S223-6 or S235.

Any one of the detergent formulations described and exemplified in EP 0342 177 also may be used in conjunction with Mutant as for detergent D3.

RESULTS Generation of Site-specific Mutations of the Subtilisin 309 Gene

Site specific mutations were performed by the method of Morinaga et al.(Biotechnology, supra). The following oligonucleotides were used forintroducing the mutations:

a) Gly 195 Glu (G195E (S001)):

A 27-mer mismatch primer, Nor-237, which also generates a novel SacIrestriction site:

5′ CACAGTATGGGCGCAGGGCTTGACATTGTCGCACCAGG 3′ Nor-237 5′GTATGGCGCAGAGCTCGACATTTGTCGC 3′

SacI

b) Arg 170 TYr (R170Y (S003)):

A 25-mer mismatch primer, Nor-577, which destroys a HaeIII site:

HaeIII

5′ GCTATCCGGCCCGTTATGCGAACGC 3′ Nor-577 3′ CGATAGGCCGTATAATACGCTTGCG 5′

c) His 120 Asp (H120D (S006)):

A 32-mer mismatch primer, Nor-735, which destroys a SphI site:

SDhI

5′ AGGGAACAATGGCATGCACGTTGCTAATTTGA 3′ Nor-735 5′AGGGAACAATGGCATGGACGTTGCTAATTTGA 3′

d) Lvs 251 Glu (K251E (S005)):

A 32-mer mismatch primer, Nor-736, which generates a XhoI site:

5′ CAAATCCGCAATCATCTAAAGAATACGGCAAC 3′ Nor-736 5′CAAATCCGCAATCATCTCGAGAATACGGCAAC 3′

XhoI

e) LYs 235 Leu (K235L (S015)):

A 24-mer mismatch primer, Nor77-856, which generates a PStI site:

5′ GCCCTTGTTAAACAAAAGAACCCA 3′ Nor-856 5′ GCCCTTGTTCTGCAGAAGAACCCA 3′

PstI

f) Arg 170 Tyr; GlY 195 Glu (R170Y;G195E (S004)):

A combination of Nor-577 and Nor-237 was performed in analogy with theabove.

g) Gly 195 Glu; Lys 251 Glu (G195E;K251E (S018)):

A combination of Nor-237 and Nor-736 was performed in analogy with theabove.

h) Arg 170 Tyr; Lvs 251 Glu (R170Y;K251E (S011)):

A combination of Nor-577 and Nor-736 was performed in analogy with theabove.

i) Ara 170 Tyr; Gly 195 Glu; Lys 251 Glu (R170Y;G195E;K251E (S012)):

A combination of Nor-577, Nor-237 and Nor-736 was performed in analogywith the above.

j) Gly 195 Glu; Lys 235 Leu (G195E;K235L):

A combination of Nor-237 and Nor-856 was performed in analogy with theabove.

k) Arg 170 Tyr; Gly 195 Glu; Lys 235 Leu (R170Y:G195E;K235L):

A combination of Nor-577, Nor-237 and Nor-856 was performed in analogywith the above.

l) His 120 Asp; Lvs 235 Leu (H120D;K235L (S016)):

A combination of Nor-735 and Nor-856 was performed in analogy with theabove.

m) His 120 Asp; Gly 195 Glu; Lys 235 Leu (H120D: G195E; K235L (S017)):

A combination of Nor-735, Nor-237 and Nor-856 was performed in analogywith the above.

n) His 120 Asp; Ara 170 Tyr; Glv 195 Glu; Lys 235 Leu (H120D; R170Y;G195E; K235L (S019)):

A combination of Nor-735, Nor-577, Nor-237 and Nor-856 was performed inanalogy with the above.

o) His 120 Asp; Arg 170 Tyr; Gly 195 Glu; Lys 235 Leu; Lys 251 Glu(H120D; R170Y; G195E: K235L; K251E (S020):

A combination of Nor-735, Nor-577, Nor-237, Nor-856 and Nor-736 wasperformed in analogy with the above.

Gapped duplex mutagenesis was performed using the plasmids pSX93, pSX119and pSX120 as templates.

pSX93 is shown in FIG. 3 and is pUC13 (Vieira, J. and Messing, J.: 1982,Gene 19: 259-268) harboring an 0.7 kb XbaI-HindIII fragment of thesubtilisin 309 gene including the terminator inserted in the polylinker.the plasmid pSX119 was used for the introduction of mutations in theN-terminal part of the enzyme. pSX119 is pUC13 harboring an EcoRI-XbaIfragment of the subtilisin 309 gene inserted into the polylinker. Thetemplates pSX93 and pSX119 thus cover the whole of the subtilisin 309gene.

Plasmid pSX120 is a plasmid where the HpaI-HindIII fragment with thesubtilisin 309 gene from pSX88 is inserted into EcoRV-HindIII on pDN1681, in a way whereby the protease gene is expressed by the amy M andamy Q promotors. pDN 1681 is obtained from pDN 1380 (Diderichsen, B. andChristiansen, L.: 1988, FEMS Microbiology Letters 56: 53-60) with aninserted 2.85 bp ClaI fragment from B. amyloliquefaciens carrying theamy Q gene with promotor (Takkinen et al.: 1983, J. Biol. Chem. 258:1007ff.). The construction of pSX120 is outlined in FIG. 1, showing thatpDN1681 is cut with EcoR5 and HindIII and pSX88 with HindIII and HpaI,whereafter ligation results in pSX120 regulated by the amy M and amy Qpromotors.

Four further plasmids pSX170, pSX172, pSX173 and pSX186 were constructedfor gapped duplex mutagenesis of the subtilisin 309 gene:

pSX170: SphI-KpnI, 700 bp from pSX120 inserted into pUC 19 SphI-KpnI,from amino acid residue 170 in mature subtilisin 309 to terminator.

pSX172: EcoRI-SphI, 1360 bp from pSX120 inserted into pUC 19 EcoRI-SphI,from the promoter to amino acid residue 170 in mature subtilisin 309.

pSX173: like pSX170, but with G195E.

pSX186: PvuII-EcoRI, 415 bp from pSX120 inserted into pUC 19HincI-EcoRI, from amino acid residue 15 to amino acid residue 206 inmature subtilisin 309.

FIG. 2 shows a somewhat detailed restriction map of pSX120 on which itis indicated which fragments were used for the construction of plasmidspSX170, pSX172, pSX173 and pSX186.

The mutation a) was performed by cutting pSX93 with XbaI and ClaI asindicated in FIG. 3 and described in the section “GENERATION OF SITEDIRECTED MUTATIONS IN THE SUBTILISIN GENE” and in unpublishedInternational Patent Application No. PCT/DK 88/00002, supra.

Mutations b), d) and e) were performed correspondingly by cutting pSX170by SphI and KpnI.

Mutations f) and g) were performed as above, but with pSX173 instead ofpSX170.

Mutation c) was performed correspondingly by cutting pSX186 by PstI andEcoRI.

The mutations h) to o) were constructed by combining DNA fragments withsingle or double mutations b) to g) using the restriction sites NheI,XbaI, ClaI, AvaII and KpnI as appropriate.

Further mutants were produced using similar methods or general methodsas known from the literature.

Subtilisin Carlsberg Mutants

For certain examples of mutations in subtilisin Carlsberg mentioned inthis specification the following changes in the nucleotide sequence ofthe gene were introduced:

Asp 14 Lys (D14K (C001)) (GAT→AAG)

Asp 120 Lys (D120K (C002)) (GAT→AAA)

Asp 140 Lys (D140K (C003)) (GAC→AAA)

Asp 14 Lys+Asp 120 Lys (D14K+D120K (C004))

Lys 27 Asp (K27D (C005)) (AAA→GAT)

Lys 27 Asp+Asp 120 Lys (K27D+D120K (C006))

Asp 172 Lys (D172K (C008)) (GAC→AAA)

Asp 14 Lys+Asp 120 Lys+Asp 140 Lys+Asp 172 Lys (D14K+D120K+D140K+D172K(C010))

Val 51 Asp (V51D (C100))

Glu 54 Thr (E54T (C101)) (GGG→ACA)

Glu 54 Tyr (E54Y (C102)) (GGG→TAT)

These changes were introduced by changing the corresponding oligos inthe fragments concerned. The correctness of the new sequences wasconfirmed after which the original oligos were replaced by these newsequences and assembled into new DNA fragments. Finally, the fragmentswere reassembled into the new subtilisin Carlsberg gene.

Expression of Mutant Subtilisins

Subsequent to sequence confirmation of the correct mutation, the mutatedDNA fragments were inserted into plasmid pSX92 or pSX120, which wereused for producing the mutants.

Plasmid pSX92 is shown in FIG. 4 and was produced by cloning the Sub 309gene into plasmid pSX62 cut at ClaI, filled in with the Klenow fragmentof DNA polymerase I and cut with HindIII prior to the insertion of thefragments DraI-NheI and NheI-HindIII from the cloned Sub 309 gene.

To express the mutants, the mutated fragments (XbaI-ClaI, XbaI-HindIII,or EcoRI-XbaI) were excised from the appropriate mutation plasmid pSX93,pSX119, pSX170, pSX172, pSX173 and pSX186, respectively, and insertedinto pSX92 or pSX120 to obtain plasmids capable of expressing thevarious mutants.

The mutated pSX92 or pSX120 was then used to transform B. subtilisDN497.

The transformed cells were then spread on LB agar plates with 10 mMphosphate, pH 7, 6 μg/ml chloramphenicol and 0.2% xylose to induce thexyn-promoter in the plasmid. The plates also contained 1% skim milk sothat the protease producing transformants could be detected by the clearhalo where the skim milk had been degraded.

After appropriate growth, the mutated enzymes were recovered andpurified.

Fermentation of the Subtilisin Carlsberg Species

In order to produce protease enzyme on the basis of the microorganismscarrying mutant genes for BPN′ as described above, a Rushton-typeChemoferm fermenter was generally used with an eight flat blade impellerand a working volume of 8 liters. The fermenter configuration wasprepared conform to the safety regulations for VMT and consisted of:

a) A pressure controller (type 4-3122, Bell & Howell) cutting off airsupply above 0.1 bar overpressure. This is done to prevent cloggedexhaust air filters.

b) A foam trap on the gas outlet made from a 20 l suction vessel havinganti-foam on the bottom.

c) A cooling water jacket without seals in order to preventcontamination of the cooling water or tapwater drain.

d) An absolute exhaust filter is used (Gelman acro 50, 0.45 micron).

e) Sampling via a sampling pump device with a small internal volume.

Controls

Gas flows were controlled using mass-flow meters (Brooks, type 5852,range 0-10 l).

The pH was controlled using a Hartmann and Braun transmitter and aPhilips controller (Witromat). Concentrated NaOH (3M) was used as aneutralizer.

Exhaust gases were analyzed using a Unor 4N (C02) and an Oxygor 7N (O2)from Maihak, Westinghouse. Oxygen tension in the medium was determinedusing an industrial polarographic sterilizable oxygen probe (Ingold type322756702).

The medium temperature was monitored using a PT100 sensor and aHoneywell temperature controller (class 84). Foaming was kept at anacceptable level using a contact electrode, while a level switchactivated an anti-foam dosage pump.

All external controls were put under the control of a Hewlett Packardmicrocomputer (HP220).

Cultivation Conditions

The inocula were prepared by incubating a shake flask culture at 30° C.for 16h at 250 rpm in a rotary shaker (LH fermentation, type MKx). 300ml inoculum was used for 8L medium being conditioned at the actualfermentation conditions (pH 7.0, 30° C., air flow 3.5 l/min, stirrer1000-1500 rpm). Dissolved oxygen concentration was kept 25% above airsaturation. Anti foaming agent used was a silicon oil based material(Rhodorsil R426, Rhone Poulenc).

Production of Subtilisin Protease

The (mutant) proteases were produced using the B. subtilis DB105 straincontaining the mutant gene as described under gene construction. Theculture medium consists of: 8 g/l NH₄Cl; 4 g/l KH₂PO₄; 4 g/l K₂HPO₄; 2g/l NaCl; 1 g/l MgSO₄.2H₂O; 10 g/l yeast extract; and 40 g/l sucrose.The pH was adjusted to 7.0, and sterilization-was performed for 45 minat 120° C. After sterilization, 25 mg/l tryptophan and 20 mg/l Neomycinwere added. Fermentations were stopped after 20-30 hours. The media werecleared from cells by centrifugation.

Proteolytic Activity of Mutant Subtilisins

The proteolytic activity of various mutants was tested against casein asprotein substrate, according to the DMC method supra. The results arepresented in Table IV.

From the table it is seen that mutant S005 exhibits a slightly enhancedactivity compared to the parent S000, whereas the remaining mutantsexhibit a slightly decreased activity.

TABLE IV Proteolytic Activity of Mutant Subtilisins Mutant RelativeActivity None (S000) 100  S001 95 S003 90 S004 85 S005 105  S006 100 S012 80 S017 90 S019 70 S020 75 S024 70

Wash Performance of Mutant Subtilisins

A:

The wash performance of various mutants in the standard liquid detergentof pH 8.14 was tested in a model system against grass juice according tothe methods detailed supra. The results are presented in table V.

TABLE V Delta R values: Enzyme Concentration Mutant 1.0 mg/l 10.0 mg/lS000 4.0 10.7 S001 5.9 12.8 S003 6.0 13.5 S004 5.8 13.0 S012 4.2  9.6S019 10.5  19.4 S020 9.4 18.6

From the table it is seen that all of the tested mutants exhibitedimproved or equal wash performance compared to the wild type parentenzyme. The wash performance of the mutants 019 and S020 is improved sothat 1.0 mg/l of these enzymes roughly stated should be able to replace10.0 mg/l of the wild type parent enzyme, thereby indicating asubstantial improvement in the wash performance for the mutant enzymesof the invention.

B:

The results from tests of some of the enzyme variants of the inventionin the modified commercial US liquid detergent at various pH values in amodel system are shown in Table VI.

TABLE VI Wash performance at different pH's Delta R pH Mutant pI_(o) 8.09.0 10.0 11.0 S000 10.02  1.4 2.6  3.1 10.1 S001 9.86 2.1 4.0  6.6 14.0S003 9.86 2.3 5.0  8.1 14.1 S004 9.68 4.1 9.7 11.7 10.9 S005 9.71 2.24.3  6.3 13.9 S012 9.09 5.7 11.9  13.8  6.3 S019 9.09 6.4 10.7  12.2 3.7 S020 6.71 7.8 10.6   8.5  2.4

The results clearly show that shifting the pI_(o) of an enzyme to the pHoptimum for the wash performance of the enzyme so that it approaches thepH of the wash liquor improves the wash performance of the enzyme.

C:

The wash performance of various mutants was tested against grass juicestained cotton cloths according to the method described in Assay A.

2.0 g/l of a liquid detergent (Detergent D3) was used. The detergent wasdissolved in ion-exchanged water. The pH was adjusted to 9.1 withNaOH/HCl.

The temperature was kept at 20° C. isothermic for 10 min.

The mutants were dosed at 0.25; 0.5; 1.0; 2.0; 5.0 and 10.0 mg enzymeprotein/l each.

The stability of the mutants was determined by measuring thedenaturation temperature (maximum excess heat capacity) by differentialscanning calorimetry, DSC. The heating rate was 0.5° C./min.

The stability was tested in a solution containing approx. 2 mg/ml of themutant in 91% standard liquid detergent, the composition of which isdescribed in Assay A. The solution was made by mixing 100 μl of enzymesolution (approx. 20 mg enzyme/ml in a buffer of 0.01 M dimethylglutaricacid, 0.002 M CaCl₂, 0.2 M H₃BO₃ and 0-0.1 M NaCl pH 6.5) with 1000 μlstandard liquid detergent.

Within the group of Subtilisin 309, mutants stability results obtainedby DSC are consistent with stability results obtained by traditionalstorage stability tests.

Results:

The wash performance of various mutants in liquid detergent is presentedin Table VII. The results are shown as improvement factors relative tothe wild type parent enzyme. The improvement factor is defined as inAssay C.

Also shown in Table VII is the denaturation temperature in standardliquid detergent by DSC and the difference between the denaturationtemperature of the wild type patent enzyme and that of the mutant inquestion.

Table VII Denaturation pI_(o) improve- Denaturation temperature calcu-ment temperature by DSC rela- mutant lated factor by DSC tive to S000S000 10.06  1   65.2   0.0 S020 7.30 7.6 58.2 −7.0 S021 9.85 1.3 69.2+4.0 S022 8.07 9.3 61.9 −3.3 S023 8.05 8.8 63.5 −1.7 S024 6.86 3.9 60.6−4.6 S025 8.94 6.7 69.1 +3.9 S035 8.07 7.0 72.5 +7.3 S201 9.85 1.4 69.4+4.2

From Table VII it is seen that all of the tested mutants exhibitimproved wash performance compared to the wild type parent enzyme. Thebest wash performance is achieved by the mutants having pI_(o) equal toor just below the pH of the wash solution.

Denaturation temperature by DSC shows that the stability of the singlemutants S021 (*36D) and S201 (N76D) is increased by 4.0° C. and 4.2° C.respectively relative to the wild type parent enzyme.

Among the mutations that are incorporated in one or more of the mutantslisted in Table VII, it has been shown that the mutations R170Y andK251E destabilize the mutant relative to the wild type parent enzyme,whereas the mutations H120D, G195E and K235L is indifferent with respectto stability.

It is seen from Table VII that mutants containing one destabilizingmutation are destabilized, even in cases, where a stabilizing mutationis included.

The stabilizing effects of *36D and N76D are additive. This is shown bythe mutants S025 and S035. S025 contains three mutations which areindifferent to stability and the stabilizing mutation *36D. Thedenaturation temperature for S025 is increased by 3.9° C. relative tothe wild type parent enzyme, which is equal to the increase measured forthe single mutant *36D, S021. S035 contains the same mutation N76D. Thedenaturation temperature for S035 is increased by 7.3° C. relative tothe wild type parent enzyme, which, within experimental error, is equalto the sum of the increase measured for the single mutants *36D, S021and N76D, S201.

D:

The wash performance of three mutants was tested against grass juicestained cotton cloth according to the method described in Assay A.

2.0 g/l of liquid detergent D3 was used. The detergent was dissolved inion-exchanged water. The pH was adjusted to 9.1 with NaOH/HCl.

The temperature was kept at 30° C. isothermic for 10 min. The mutantswere dosed at 1.0 and 10.0 mg enzyme protein/l each.

Results:

The wash performance of three mutants in commercial US-liquid detergentwas tested against grass juice. The results are shown in Table VIII.

TABLE VIII Delta R values: calculated Enzyme concentration Mutant pI_(o)1.0 mg/l 10.0 mg/l S000 10.06  4.5 13.6 S003 9.75 9.4 18.0 S004 9.5413.7  18.1 S006 9.85 6.0 15.6

From Table VIII it is seen that all of the mutants exhibit improved washperformance relative to the wild type parent enzyme. It is further seenthat the best performance is achieved by the mutant having PI_(o)closest to the pH of the wash solution.

E:

The wash performance of two mutants was tested against grass juicestained cotton cloth according to the conditions described in Example D.

Results

The wash performance of two mutants in detergent D3 was tested againstgrass juice stained cotton cloth. The results are shown in Table IX.

TABLE IX Delta R values: calculated Enzyme concentration Mutant pI_(o)1.0 mg/l 10.0 mg/l S000 10.06  5.8 15.3 S015 9.95 8.4 20.0 S017 9.4017.0  20.8

From Table IX it is seen that all of the mutants exhibit improved washperformance relative to the wild type parent enzyme. It is further seenthat the best performance is achieved by the mutant having pI_(o)closest to the pH of the wash solution.

F:

The wash performance of various mutants was tested on grass juicestained cotton cloth according to the method described in Assay A.

2.0 g/l of detergent D3 was used.

The detergent was dissolved in buffer (0.0025 M Boric acid and 0.001 Mdisodium hydrogen phosphate prepared in ion exchanged water). The pH wasadjusted to 7.0, 8.0, 9.0 and 10.0 respectively with NaOH/HCl. Thetemperature was kept at 30° C. isothermic for 10 min.

The mutants were dosed at 0.2 mg enzyme protein/l each.

Results:

The wash performance of some of the enzyme variants of the invention atvarious pH values in a model system are shown in table X.

TABLE X Delta R Vari- pH ant Mutation pI_(o) 7.0 8.0 9.0 10.0 S00010.06  0.6 0.8 4.4 7.0 S015 K235L 9.95 1.3 2.4 6.0 8.8 S021 *36D 9.852.1 3.2 5.6 8.3 S017 H120D,G195E,K235L 9.40 2.9 5.4 10.8  14.1  S025*36D,H120D,R170Y, 8.95 4.3 9.5 13.9  13.1  K235L S023 *36D,H120D,R170Y,8.05 9.6 13.0  12.4  9.2 G195E,K235L S024 *36D,H120D,R170Y, 6.86 9.410.4  6.7 4.8 G195E,K235L,K251E

The results in Table X clearly show that shifting the pI_(o) of aprotease towards the pH of the wash liquor improves the wash performanceof the protease.

The results also show that all variants tested have improved performancecompared to the wild type parent enzyme at pH below 10.0.

G:

The wash performance of various mutants was tested on grass juicestained cotton cloths according to the method described in Assay A.

2.0 g/l of liquid detergent D3 was used. The detergent was dissolved in0.005 M glycine prepared in ion-exchanged water). The pH was adjusted to10.0, 10.25, 10.50, 10.75, 11.0, 11.5 and 12.0, respectively, with NaOH.The temperature was kept at 30° C. isothermic for 10 minutes.

The mutants were dosed at 0.2 mg enzyme protein/l each.

Results:

The wash performance of some of the enzyme variants of the invention atvarious pH values in a model system are shown in table XI. In this casevariants with slightly higher PI_(o) than the wild type parent enzymewas investigated. The pH range from 10.0 to 12.0 is investigated in moredetails than in prior examples.

TABLE XI Delta R pH Variant Mutation pI_(o) 10.0 10.25 10.50 S000 10.067.0 8.7 10.5  S027 E895 10.28 6.0 8.5 9.8 S028 D181N 10.28 6.9 9.8 10.6 S032 D197N 10.28 4.7 9.2 10.8  S033 E271Q 10.28 7.1 6.7 7.8 S031D197N,E271Q 10.53 4.7 7.2 7.0 Delta R pH Variant Mutation pI_(o) 10.7511.0 11.5 12.0 S000 10.06 12.5 14.4 10.6 3.8 S027 E895 10.28 11.9 14.312.8 5.0 S028 D181N 10.28 13.0 14.4 10.7 4.6 S032 D197N 10.28 13.8 13.511.3 5.0 S033 E271Q 10.28 10.4 13.7 13.3 6.3 S031 D197N,E271Q 10.53 10.713.0 14.4 8.7

The data in Table XI show that at high pH values maximum performance isachieved at pH values a little above the calculated pI_(o). Stillincreasing the pI_(o) of the protease tends to increase the pH ofmaximum performance. The effects are not as pronounced as it is seen atlow pH values (assay B and G).

H:

In order to visualize the correlation between isoelectric point of theprotease and the pH at which the protease has its maximum performance,the results from examples B, F and G are used to find the pH at whicheach of the investigated variants (and the wild type parent enzyme) hasits maximum performance. In FIG. 5 this pH_(max) is shown as a functionof the calculated pI₀.

Taking into account that the pH range is investigated in steps of 1.0 pHvalue the correlation is obvious.

Concerning the combination of the mutants of the invention with lipaseexperimental results led to the following practical conclusions:

Lipase was stable for an hour in wash liquor of type O at 37° C. Thepresence of Savinase® led to rapid deactivation. Kazusase® led tosubstantially less inactivation of lipase over the period of the test.

Proteinase K was seen to be less aggressive to lipase than Savinase®,but more so than Kazusase®. Subtilisin BPN′ did not however inactivatelipase at all under these conditions.

Preferred proteases for use e.g. in connection with lipase in washcompositions represented by type O, are mutants S001, S003, S004, S012,S019, S020, S021, S025, S035 and S235.

Type O wash liquor was a 5 g/l solution at 37° C. derived from thefollowing detergent formulation (% by wt):

anionic surfactant 6   nonionic surfactant 5   fatty acid 2.8 acrylicpolymer 3   zeolite 22   carbonate 10   sulphate 17.5  clay 8   tertiaryamine 2   perborate monohydrate 13   minors and water to 100.

Preferred proteases for use in connection with lipase in washcompositions represented by type W are mutant S020, S021, S025, S035 andS235.

Type W wash liquor was a 2 g/l solution of a liquid detergent having thefollowing formulation (% by wt):

anionic surfactant 16   nonionic surfactant 7   hydrotrope 6   citricacid 6.5 NaOH 4.1 monoethanolamine 2   minors and water to 100.

Detergent compositions Comprising Lipase

Experimental tests of lipase stability were carried out for exampleusing the following materials:

1 LU/ml Pseudomonas cepacia lipase was incubated in wash liquor of eachof two types, O and W (described below). Aliquots were taken atintervals and tested for lipase activity. Parallel incubations werecarried out without protease or with protease on the retention of lipaseactivity. Wild-type proteases were tested at 20 GU/ml, mutated proteaseswere tested at 015 microgram/ml.

Experimental results led to the following conclusions:

Lipase was stable for an hour in wash liquor of type 0 at 37° C. Thepresence of Savinase™ led to rapid deactivation. Kazusase™ led tosubstantially less inactivation of lipase over the period of the test.

Proteinase K was seen to be less aggressive to lipase than Savinase, butmore so than Kazusase. Subtilisin BPN′ did not however inactivate lipaseat all under these conditions.

Preferred proteases for use e.g. in connection with lipase in washcompositions represented by type O, are Mutants S001, S003, S004, S012,S019 and S020.

Type O wash liquor was a 5 g/l solution at 37° C. derived from thefollowing detergent formulation (wt %):

anionic surfactant 6   nonionic surfactant 5   fatty acid 2.8 acrylicpolymer 3   zeolite 22   carbonate 10   sulphate 17.5  clay 8   tertiaryamine 2   perborate monohydrate 13   minors and water to 100.

A preferred protease for use e.g. in connection with lipase in washcompositions represented by type W is example S020. Type W wash liquorwas a 2 g/l solution of a liquid detergent having the followingformulation (wt %):

anionic surfactant 16   nonionic surfactant 7   hydrotrope 6   citricacid 6.5 NaOH 4.1 monoethanolamine 2   minors and water to 100.

The invention is illustrated by way of the following non-limitingExamples:

Example 1L

A detergent powder is formulated to contain: total active detergentabout 16%, anionic detergent about 9%, nonionic detergent about 6%,phosphate-containing builder about 20%, acrylic or equivalent polymerabout 3.5%, perborate or peracid bleach precursor about 6-18%,amino-containing bleach activator about 2%, silicate or otherstructurant about 3.5%, protease enzyme about 8 glycine units/mg, withalkali to adjust to desired pH in use, and neutral inorganic salt, andenzymes (about 0.5% each protease and lipase).

The anionic detergent is a mixture of sodium dodecylbenzene sulphonate6% and primary alkyl sulphate 3%. The nonionic detergent is anethoxylate of an approx. C13-C15 primary alcohol with 7 ethoxylateresidues per mole. The phosphate builder is sodium tripolyphosphate. Thepolymer is polyacrylic acid. The perborate or peracid bleach precursoris sodium tetraborate tetrahydrate or monohydrate. The activator istetraacetylethylenediamine. The structurant is sodium silicate. Theneutral inorganic salt is sodium sulphate. The enzymes comprise lipaseand Mutant S001 as the protease. Alternatively, the protease enzyme isselected from S003, S004, C002, C003 and C004.

Example 2L

A detergent powder is formulated to contain: total active detergentabout 16%, anionic detergent about 9%, nonionic detergent about 6%,zeolite-containing builder about 20%, acrylic or equivalent polymerabout 3.5%, perborate or peracid bleach precursor about 6-18%,amino-containing bleach activator about 2%, silicate or otherstructurant about 3.5%, protease enzyme about 8 glycine units/mg, withalkali to adjust to desired pH in use, and neutral inorganic salt, andenzymes (about 0.5% each of protease and lipase enzyme).

The anionic detergent is a mixture of sodium dodecylbenzene sulphonate6% and primary alkyl sulphate 3%. The nonionic detergent is anethoxylate of an approx. C13-C15 primary alcohol with 7 ethoxylateresidues per mole. The zeolite builder is type A zeolite. The polymer ispolyacrylic acid. The perborate bleach precursor is sodium tetraboratetetrahydrate or monohydrate. The activator istetraacetylethylenediamine. The structurant is sodium silicate. Theneutral inorganic salt is sodium sulphate. The protease enzyme is MutantS001. Alternatively, the enzyme is selected from S003, S004, C002, C003and C004. Lipase and protease are both present at about 0.5%.

Example 3L

An aqueous detergent liquid is formulated to contain:Dodecylbenzene-sulphonic acid 16%, C12-C15 linear alcohol condensed with7 mol/mol ethylene oxide 7%, monoethanolamine 2%, citric acid 6.5%,sodium xylenesulphonate 6%, sodium hydroxide about 4.1%, protease 0.5%,minors and water to 100%. The pH is adjusted to a value between 9 and10. The protease enzyme is Mutant S0020. Alternatively, the proteaseenzyme is selected from S0019, S0012, S001, S003 and S004. Lipase andprotease are both present at about 0.5%.

Example 4L

A nonaqueous detergent liquid is formulated using 38.5% C13-C15 linearprimary alcohol alkoxylated with 4.9 mol/mol ethylene oxide and 2.7mol/mol propylene oxide, 5% triacetin, 30% sodium triphosphate, 4% sodaash, 15.5% sodium perborate monohydrate containing a minor proportion ofoxoborate, 4% TAED, 0.25% EDTA of which 0.1% as phosphonic acid, Aerosil0.6%, SCMC 1%, and 0.6% protease. The pH is adjusted to a value between9 and 10, e.g. about 9.8. The protease enzyme is Mutant S001, S003 orS004. Lipase and protease are both present at about 0.5%.

Example 5L

A detergent powder is formulated in the form of a granulate having abulk density of at least 600 g/l, containing about 20% by weightsurfactant of which about 10% is sodium dodecylbenzene sulphonate, andthe remainder is a mixture of Synperonic A7 and Synperonic A3 (about5.5% to 4.5%), and zero neutral inorganic salt (e.g. sodium sulphate),plus phosphate builder about 33%, sodium perborate tetrahydrate about16%, TAED activator about 4.5%, sodium silicate about 6%, and minorsincluding sodium carbonate about 2%, and moisture content about 10%.Enzymes (about 0.5% each of lipase and protease enzyme) are included.The protease enzyme is Mutant S001. Alternatively, the protease enzymeis selected from S003, S004, C002, C003 and C004.

Example 6L

A detergent powder is formulated in the form of a granulate having abulk density of at least 600 g/l, containing about 20% by weightsurfactant of which about 9% is sodium dodecylbenzene sulphonate, andthe remainder is a mixture of Synperonic A7 and Synperonic A3(respectively about 5% and 6%), and zero neutral inorganic salt (e.g.sodium sulphate), plus zeolite builder about 30%, sodium perboratetetrahydrate about 14%, TAED activator about 3.6%, and minors includingsodium carbonate about 9%, Dequest 2047™ about 0.7%, and moisturecontent about 10%. Enzymes (about 0.5% each of lipase and proteaseenzyme) are included. The protease enzyme is Mutant S001. Alternatively,the protease enzyme is selected from S003, S004, C002, C003 and C004.

Example 7L

A detergent powder is formulated to contain: Dodecylbenzenesulphonicacid 6%, C12-C15 linear alcohol condensed with 7 mol/mol ethylene oxide5%, fatty acid soap 3%, Sokolan CP5 polymer™ 3%, zeolite A 22%, sodiumcarbonate 10%, sodium sulphate 17%, clay particles 8%, sodium perboratetetrahydrate 13%, tetraacetyl-ethylenediamine 2%, protease 0.5%, minorsand water to 100%. The pH is adjusted to a value between 9 and 10. Theprotease enzyme is Mutant S020. Alternatively, the enzyme is selectedfrom S019, S012, S004, S001 and S003. Lipase and protease are bothpresent at about 0.5%.

Example 8L

A detergent (soap) bar is formulated as follows: soap based onpan-saponified 82% tallow, 18% coconut oil, neutralized with 0.15%orthophosphoric acid, mixed with protease (about 8 GU/mg of the barcomposition) and with sodium formate 2%, borax 2%, propylene glycol 2%and sodium sulphate 1%, is then plodded on a soap production line. Theprotease enzyme is Mutant S001. Alternatively, the protease is selectedfrom S003, S004, C002, C003 and C004. Lipase and protease are bothpresent at about 0.5%.

In further embodiments of the invention, structured liquid detergentscan further contain e.g. 2-15% nonionic surfactant, 5-40% totalsurfactant, comprising nonionic and optionally anionic surfactant, 5-35%phosphate-containing or non-phosphate-containing builder, 0.2-0.8%polymeric thickener, e.g. cross-linked acrylic polymer with m.w. over10⁶, at least 10% sodium silicate, e.g. as neutral waterglass, alkali(e.g. potassium-containing alkali) to adjust to desired pH, preferablyin the range 9-10 or upwards, e.g. about pH 11, with a ratio sodiumcation: silicate anion (as free silica) (by weight) less than 0.7:1, andviscosity of 0.3-30 Pa.s (at 20° C. and 20 reciprocal secs).

For example, such detergents can contain about 5% nonionic surfactantC13-15 alcohol alkoxylated with about 5 EO groups and about 2.7 POgroups per mole, 15-23% neutral waterglass with 3.5 weight ratio betweensilica and sodium oxide, 13-19% KOH, 8-23% STPP, 0-11% sodium carbonate,0.5% Carbopol 941™. Protease may be incorporated at for example 0.5% ofMutant S001.

Although the present invention has been discussed and exemplified inconnection with various specific embodiments thereof this is not to beconstrued as a limitation to the applicability and scope of thedisclosure, which extends to all combinations and subcombinations offeatures mentioned and described in the foregoing as well as in theattached patent claims.

What is claimed is:
 1. A modified subtilisin comprising a substitutionof an amino acid with a different amino acid at one or more positionsselected from the group consisting of: 27, 105, 106, 141, 251, 256, and257, wherein each position corresponds to the position of the amino acidsequence of the mature subtilisin BPN′.
 2. The modified subtilisin ofclaim 1, which comprises a substitution at position
 27. 3. The modifiedsubtilisin of claim 1, which comprises a substitution at position 105.4. The modified subtilisin of claim 1, which comprises a substitution atposition
 106. 5. The modified subtilisin of claim 1, which comprises asubstitution at position
 141. 6. The modified subtilisin of claim 1,which comprises a substitution at position
 251. 7. The modifiedsubtilisin of claim 1, which comprises a substitution at position 256.8. The modified subtilisin of claim 1, which comprises a substitution atposition
 257. 9. The modified subtilisin of claim 1, wherein thesubtilisin that is modified is selected from the group consisting ofsubtilisin BPN′, subtilisin amylosacchariticus, subtilisin 168,subtilisin mesentericopeptidase, subtilisin Carlsberg, subtilisin DY,subtilisin 309, subtilisin 147, thermitase, aqualysin, Bacillus PB92protease, proteinase K, Protease TW7 and Protease TW3.
 10. The modifiedsubtilisin of claim 9, wherein the subtilisin that is modified issubtilisin
 309. 11. The modified subtilisin of claim 1, which has a netelectrostatic charge that is different from that of the parentsubtilisin at the same pH and has an isoelectric point that is higher orlower than that of the parent subtilisin.
 12. The modified subtilisin ofclaim 11, which has a net electrostatic charge that is more positivethan that of the parent subtilisin.
 13. The modified subtilisin of claim11, which has a net electrostatic charge that is more negative than thatof the parent subtilisin.
 14. The modified subtilisin of claim 1, whichhas at least one different amino acid that is more positive than theamino acid in the corresponding position in the parent subtilisin. 15.The modified subtilisin of claim 1, which has at least one differentamino acid that is more negative than the amino acid in thecorresponding position of the parent subtilisin.
 16. A detergentcomposition comprising a modified subtilisin of claim 1 and asurfactant.
 17. A modified subtilisin comprising an insertion of anamino acid at position 36, wherein position 36 corresponds to theposition of the amino acid sequence of the mature subtilisin BPN′. 18.The modified subtilisin of claim 17, wherein the amino acid is anegatively charged amino acid.
 19. The modified subtilisin of claim 18,wherein the amino acid is D or E.
 20. The modified subtilisin of claim17, wherein the amino acid is a neutral or positively (harged aminoacid.
 21. The modified subtilisin of claim 20, wherein the amino acid isA, K, N, Q, or R.
 22. A detergent composition comprising a modifiedsubtilisin of claim 17 and a surfactant.
 23. A modified subtilisincomprising a mutation in an amino acid sequence of a subtilisin at aposition numbered according to the amino acid sequence of the maturesubtilisin BPN′, wherein the mutation is selected from the groupconsisting of: Q12K, Q12R, R19Q, T22R, D32*, T38K, T38R, R45A,E53G+K235L, E53K, E53R, E54G, E54T, E54Y, Q59E, T71D, T71D+G195E, E89S,G97D, G97D+H120K, N97D, N97D+S98D, N97D+T213D, A98K, A98R, S98D,S98D+T213D, S99D, S99D+N140K, D120K, H120D+G195E,H120D+G195E+K235L+K251E, H120D+R170Y+G195E+K235L+K251E, H120K, P129D,D140K, N140K, N140R, S156E, A158K, A158R, Y167V, R170Y+K251E, Y171T,D172K, R186P G195E+T213R, G195F, D197E, D197K, D197N, Q206D, Q206E,T213D, Y214S, Y214T, K235L, K235R, K237R, W241L, and S265R.
 24. Amodified subtilisin of claim 23, wherein the mutation comprises Q12K.25. A modified subtilisin of claim 23, wherein the mutation comprisesQ12R.
 26. A modified subtilisin of claim 23, wherein the mutationcomprises R19Q.
 27. A modified subtilisin of claim 23, wherein themutation comprises T22R.
 28. A modified subtilisin of claim 23, whereinthe mutation comprises D32*.
 29. A modified subtilisin of claim 23,wherein the mutation comprises T38K.
 30. A modified subtilisin of claim23, wherein the mutation comprises T38R.
 31. A modified subtilisin ofclaim 23, wherein the mutation comprises R45A.
 32. A modified subtilisinof claim 23, wherein in the mutation comprises E53G+K235L.
 33. Amodified subtilisin of claim 23, wherein the mutation comprises E53K.34. A modified subtilisin of claim 23, wherein the mutation comprisesE53R.
 35. A modified subtilisin of claim 23, wherein the mutationcomprises E54G.
 36. A modified subtilisin of claim 23, wherein themutation comprises E54T.
 37. A modified subtilisin of claim 23, whereinthe mutation comprises E54Y.
 38. A modified subtilisin of claim 23,wherein the mutation comprises E59E.
 39. A modified subtilisin of claim23, wherein the mutation comprises T71D.
 40. A modified subtilisin ofclaim 23, wherein the mutation comprises T71D+G195E.
 41. A modifiedsubtilisin of claim 23, wherein the mutation comprises E89S.
 42. Amodified subtilisin of claim 23, wherein the mutation comprises G97D.43. A modified subtilisin of claim 23, wherein the mutation comprisesG97D+H120K.
 44. A modified subtilisin of claim 23, wherein the mutationcomprises N97D.
 45. A modified subtilisin of claim 23, wherein themutation comprises N97D+S98D.
 46. A modified subtilisin of claim 23,wherein the mutation comprises N97D+T213D.
 47. A modified subtilisin ofclaim 23, wherein the mutation comprises A98K.
 48. A modified subtilisinof claim 23, wherein the mutation comprises A98R.
 49. A modifiedsubtilisin of claim 23, wherein the mutation comprises S98D.
 50. Amodified subtilisin of claim 23, wherein the mutation comprisesS98D+T213D.
 51. A modified subtilisin of claim 23, wherein the mutationcomprises S99D.
 52. A modified subtilisin of claim 23, wherein themutation comprises S99D+N140K.
 53. A modified subtilisin of claim 23,wherein the mutation comprises D120K.
 54. A modified subtilisin of claim23, wherein the mutation comprises H120+G195E.
 55. A modified subtilisinof claim 23, wherein the mutation comprises H120D+R195E+K235L+K251E. 56.A modified subtilisin of claim 23, wherein the mutation comprisesH120D+R170Y+G195E+K235L+K251E.
 57. A modified subtilisin of claim 23,wherein the mutation comprises N120K.
 58. A modified subtilisin of claim23, wherein the mutation comprises P129D.
 59. A modified subtilisin ofclaim 23, wherein the mutation comprises D140K.
 60. A modifiedsubtilisin of claim 23, wherein the mutation comprises N140K.
 61. Amodified subtilisin of claim 23, wherein the mutation comprises N140R.62. A modified subtilisin of claim 23, wherein the mutation comprisesS156E.
 63. A modified subtilisin of claim 23, wherein the mutationcomprises A158K.
 64. A modified subtilisin of claim 23, wherein themutation comprises A158R.
 65. A modified subtilisin of claim 23, whereinthe mutation comprises Y167V.
 66. A modified subtilisin of claim 23,wherein the mutation comprises R170Y+K251E.
 67. A modified subtilisin ofclaim 23, wherein the mutation comprises R171T.
 68. A modifiedsubtilisin of claim 23, wherein the mutation comprises D172K.
 69. Amodified subtilisin of claim 23, wherein the mutation comprises D186P.70. A modified subtilisin of claim 23, wherein the mutation comprisesG195E+T213R.
 71. A modified subtilisin of claim 23, wherein the mutationcomprises G195F.
 72. A modified subtilisin of claim 23, wherein themutation comprises G197E.
 73. A modified subtilisin of claim 23, whereinthe mutation comprises D197K.
 74. A modified subtilisin of claim 23,wherein the mutation comprises D197N.
 75. A modified subtilisin of claim23, wherein the mutation comprises Q206D.
 76. A modified subtilisin ofclaim 23, wherein the mutation comprises Q206E.
 77. A modifiedsubtilisin of claim 23, wherein the mutation comprises T213D.
 78. Amodified subtilisin of claim 23, wherein the mutation comprises Y214S.79. A modified subtilisin of claim 23, wherein the mutation comprisesY214T.
 80. A modified subtilisin of claim 23, wherein the mutationcomprises K235L.
 81. A modified subtilisin of claim 23, wherein themutation comprises K235R.
 82. A modified subtilisin of claim 23, whereinthe mutation comprises K237R.
 83. A modified subtilisin of claim 23,wherein the mutation comprises K241L.
 84. A modified subtilisin of claim23, wherein the mutation comprises S265R.
 85. The modified subtilisin ofclaim 23, wherein the subtilisin that is modified is selected from thegroup consisting of subtilisin BPN′, subtilisin amylosacchariticus,subtilisin 168, subtilisin mesentericopeptidase, subtilisin Carlsberg,subtilisin DY, subtilisin 309, subtilisin 147, thermitase, aqualysin,Bacillus PB92 protease, proteinase K, Protease TW7 and Protease TW3. 86.The modified subtilisin of claim 85, wherein the subtilisin that ismodified is subtilisin
 309. 87. A detergent composition comprising amodified subtilisin of claim 23 and a surfactant.